MicroRNA-455-3p as a Peripheral Biomarker for Alzheimer&#39;s Disease

ABSTRACT

The present invention includes a method of protecting neuronal cells from Aβ-induced toxicities in a subject comprising: providing the subject in need of treatment for an Aβ-induced toxicity with an agent that increases the miRNA-445-3p in the subject, wherein an increase in miR-455-3p expression in the neuronal cells enhances neuronal cell survival.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/630,068 filed Jan. 10, 2020, which is a NationalStage of International Application No. of PCT/US2018/041840, filed onJul. 12, 2018 claiming the priority of U.S. Provisional Application No.62/531,760, filed on Jul. 12, 2017, the content of each of which isincorporated by reference herein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the treatment of Alzheimer'sDisease.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

The present application includes a Sequence Listing, which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 12, 2018, isnamed TECH2106WO_SeqList and is 3 kilobytes in size.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with biomarkers for Alzheimer's Disease.

One such invention is taught in U.S. Pat. No. 9,188,595, issued to Zhao,et al., and entitled “Alzheimer's disease diagnosis based onmitogen-activated protein kinase phosphorylation.” Briefly, theseinventors teach a method of diagnosing Alzheimer's disease in a patientby determining whether the phosphorylation level of an indicator proteinin cells of the patient after stimulus with an activator compound isabnormally elevated as compared to a basal phosphorylation level, theindicator protein being e.g. Erk1/2 and the activator compound isbradykinin.

Another invention is taught in United States Patent Publication No.20140378439, filed by Dezso, et al., entitled “MicroRNA BiomarkersIndicative Of Alzheimer's Disease.” These inventors are said to teach amethod of diagnosing Alzheimer's Disease in a subject, by determiningthe level of at least one miRNA in a sample derived from the subject,wherein a change in the level of the at least one miRNA relative to asuitable control is indicative of Alzheimer's Disease in the subject.Methods for monitoring the course of Alzheimer's Disease, methods oftreating a subject having Alzheimer's Disease, and kits for diagnosingAlzheimer's Disease are also said to be taught.

Yet another invention is taught in United States Patent Publication No.20140302068, filed by Khoo, et al., entitled “MicroRNA Biomarkers forDiagnosing Parkinson's Disease”, which is said to teach theidentification, development and validation of plasma-based circulatingmicroRNA (miRNAs) biomarkers useful in determining if a subject hasParkinson's disease (PD), is at increased risk of developing PD, or hasPD that is progressing or is in remission.

Another such invention is taught in United States Patent Publication No.20140031245, filed by Khan, et al., and entitled “Alzheimer'sDisease-Specific Alterations Of The Erk1/Erk2 PhosphorylationRatio-Alzheimer's Disease-Specific Molecular Biomarkers (ADSMB)”.Briefly, these applicants are said to teach methods of diagnosingAlzheimer's Disease as well as to methods of confirming the presence orabsence of Alzheimer's Disease in a subject. These application methodsof identifying a lead compound useful for the treatment of Alzheimer'sDisease by contacting non-Alzheimer's cells with an amyloid betapeptide, stimulating the cells with a protein kinase C activator,contacting the cells with a test compound, and determining the value ofan Alzheimer's Disease-specific molecular biomarker. The invention isalso said to be directed to kits containing reagents for the detectionand diagnosis of the presence or absence of Alzheimer's Disease usingthe Alzheimer's Disease-specific molecular biomarkers disclosed.

Yet another invention is taught in International Patent Publication No.WO2009009457A1, filed by Wang, et al., entitled “Alzheimer'sdisease-specific micro-RNA microarray and related methods.” Theseinventors are said to disclosed the diagnosis and/or prognosis ofAlzheimer's disease in subjects by measuring amounts of one or moremicro-RNAs correlated with Alzheimer's disease present in a biologicalsample, including blood for example, from a subject.

Despite the prior art disclosures, compositions and methods to detectAlzheimer's disease (AD) early, before clinical symptoms develop, areurgently needed to intervene as soon as possible in disease progression.Also needed are early peripheral microRNA (miRNA) biomarkers for AD.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method of protectingneuronal cells from Aβ-induced toxicities in a subject comprising:providing the subject in need of treatment for an Aβ-induced toxicitywith an agent that increases the miRNA-445-3p in the subject, wherein anincrease in miR-455-3p expression in the neuronal cells enhancesneuronal cell survival. In one aspect, the method comprises agent thatincreases the miRNA-445-3p in the subject is a nucleic acid vector thatexpresses miRNA-445-3p in neuronal cells of the subject. In anotheraspect, the agent enhances at least one of: mitochondrial biogenesis,enhances synaptic activity or mitochondrial health. In another aspect,the method comprises providing the subject with a cyclooxygenaseinhibitor, a Catecholamine transferase inhibitor, a protein kinasesinhibitor, a Neurotransmitter transporter inhibitor, a Renin-angiotensinsystem inhibitor, a EGFR tyrosine kinase inhibitor, or a HMG-CoAreductase inhibitor. In another aspect, the method comprises identifyinga subject in need of treatment for the Aβ-induced toxicity by detectingthe presence or the increase in miRNA-445-3p to produce a score that isindicative of a likelihood of developing AD, wherein a higher scorerelative to a healthy control indicates that the patient is likely tohave the prognosis for transitioning to classified AD, wherein thehealthy control is derived from a non-AD patient with no clinicalevidence of AD. In another aspect, the patient is identified at least0.1, 0.9, 2.0, 3.5, or greater than 3.5 years prior to reaching clinicaldisease classification. In another aspect, the method comprisesassessing comprises RT-PCR, qRT-PCR, biochip, singleplexed ormultiplexed RT-PCR. In another aspect, the method comprises assessing atleast one additional biomarker selected from: hsa-miR-3613-3p andhsa-miR-4668-5p, which is up-regulated. In another aspect, the methodcomprises assessing at least one additional biomarker selected from:hsa-mir-320d-2, hsa-miR-378h, hsa-miR-3921, hsa-miR-6805-5p,hsa-miR-92a-3p, hsa-miR-3613-5p, which is down-regulated. In anotheraspect, the method comprises obtaining the dataset associated with thesample comprises obtaining the sample and processing the sample toexperimentally determine the dataset, or wherein obtaining the datasetassociated with the sample comprises receiving the dataset from a thirdparty that has processed the sample to experimentally determine thedataset. In another aspect, the method comprises identifying a relativeof the patient at risk for AD by obtaining a score from a datasetassociated with a blood, serum, or plasma sample from a relative of theAD patient prior to reaching clinical disease classification. In anotheraspect, the healthy control is a pre-determined average level derivedfrom a healthy individual with no clinically documented evidence of AD.In another aspect, at least one of: the miR-455-3p has a greater that20-fold increase in expression in Braak stage V and VI compared tocontrols; the expression of miR-3613-3p is higher in the brain tissuesat Braak stage V when compared to controls; the expression ofmiR-4668-5p up-regulated in AD brains at Braak stages IV, V, and VI, butless than miR-455-3p or MiR-4674; the expression of mir-6722 wasdown-regulated in AD serum samples Braak stages I to III, but increasedin the AD patients at Braak stage VI, V, and VI; the upregulation ofmiR-455-3p was significantly higher in postmortem brains from ADpatients at Braak stage V having an ApoE (3/4) genotype when compared tocontrols; or the Braak stage can be differentiated between Stage I toIII versus Brask Stage IV to VI by comparing the expression ofmiR-455-3p, miR-3613-3p, miR-4668-5p, and mir-6722.

In another embodiment, the present invention includes a method fortreating at least one of amyloid precursor protein (APP) processing,amyloid beta (Aβ) formation, defective mitochondrial biogenesis/dynamicsand synaptic damage in AD progression in a subject suspected of havingAlzheimer's disease (AD) comprising: providing the subject in need oftreatment for an Aβ-induced toxicity with a vector increases theexpression of miRNA-445-3p in the subject, wherein an increase inmiR-455-3p expression in the neuronal cells enhances neuronal cellsurvival, wherein the amount is sufficient to at least one of correctamyloid precursor protein (APP) processing, prevent amyloid beta (Aβ)formation, decrease defective mitochondrial biogenesis/dynamics orreduce or prevent synaptic damage in AD progression. In another aspect,the expression of miRNA-445-3p in neuronal cells reduces a level ofmutant APP, Aβ(1-40) and Aβ(1-42), and C99 by miR-455-3p. In anotheraspect, the method comprises administering a cyclooxygenase inhibitor, aCatecholamine transferase inhibitor, a protein kinases inhibitor, aNeurotransmitter transporter inhibitor, a Renin-angiotensin systeminhibitor, a EGFR tyrosine kinase inhibitor, or a HMG-CoA reductaseinhibitor. In another aspect, the method comprises assessing at leastone additional biomarker selected from: hsa-miR-3613-3p andhsa-miR-4668-5p, which is up-regulated. In another aspect, the methodcomprises assessing at least one additional biomarker selected from:hsa-mir-320d-2, hsa-miR-378h, hsa-miR-3921, hsa-miR-6805-5p,hsa-miR-92a-3p, hsa-miR-3613-5p, which is down-regulated. In anotheraspect, the patient is identified at least 0.1, 0.9, 2.0, 3.5, orgreater than 3.5 years prior to reaching clinical diseaseclassification. In another aspect, the step of assessing comprisesRT-PCR, qRT-PCR, biochip, singleplexed or multiplexed RT-PCR.

In another embodiment, the present invention includes a method fortreating a subject with Alzheimer's Disease (AD) comprising: obtaining ablood, serum, or plasma sample from the AD patient; assessing thedataset for a presence or an increase in an amount of miRNA-445-3p; andif the subject has a decrease in miRNA-445-3p expression or is in needof an increase in the expression of miRNA-445-3p, providing the subjectin need of treatment for an Aβ-induced toxicity an agent that increasesthe miRNA-445-3p in the subject, wherein an increase in miR-455-3pexpression in the neuronal cells enhances neuronal cell survival. Inanother aspect, the method comprises administering a cyclooxygenaseinhibitor, a Catecholamine transferase inhibitor, a protein kinasesinhibitor, a Neurotransmitter transporter inhibitor, a Renin-angiotensinsystem inhibitor, a EGFR tyrosine kinase inhibitor, or a HMG-CoAreductase inhibitor. In another aspect, the step of assessing comprisesRT-PCR, qRT-PCR, biochip, singleplexed or multiplexed RT-PCR. In anotheraspect, the method comprises assessing at least one additional biomarkerselected from: hsa-miR-3613-3p and hsa-miR-4668-5p, which isup-regulated. In another aspect, the method further comprises assessingat least one additional biomarker selected from: hsa-mir-320d-2,hsa-miR-378h, hsa-miR-3921, hsa-miR-6805-5p, hsa-miR-92a-3p,hsa-miR-3613-5p, which is down-regulated. In another aspect, the datasetassociated with the sample comprises obtaining the sample and processingthe sample to experimentally determine the dataset, or wherein obtainingthe dataset associated with the sample comprises receiving the datasetfrom a third party that has processed the sample to experimentallydetermine the dataset. In another aspect, the method further comprisesidentifying a relative of the patient at risk for AD by obtaining ascore from a dataset associated with a blood, serum, or plasma samplefrom a relative of the AD patient prior to reaching clinical diseaseclassification. In another aspect, the healthy control is apre-determined average level derived from a healthy individual with noclinically documented evidence of AD.

In another embodiment, the present invention includes a compositioncomprising a recombinant anti-miRNA-445-3p nucleic acid sufficient toknockdown the expression of miRNA-445-3p.

In another embodiment, the present invention includes a method oftreating a subject in need of therapy for Alzheimer's Diseasecomprising: identifying a subject suspected of having Alzheimer'sDisease; and providing the subject with an effective amount of arecombinant miRNA-445-3p expression vector sufficient to upregulate theexpression of miRNA-445-3p in the subject in need of therapy forAlzheimer's Disease.

DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 is a heat map showing hierarchical clustering of miRNAs in ADpatients and healthy controls. Left side showed the Transcript clusterID of differentially expressed 7 miRNAs. Red and green color indicatedhigh and low expression intensities.

FIGS. 2A to 2E show qRT-PCR validation of serum samples. Expression of(FIG. 2A) miR-455-3p, (FIG. 2B) miR-4668-5p, (FIG. 2C) miR-4674, (FIG.2D) miR-3613-3p and (FIG. 2E) mir-6722 in healthy controls, MCI subjectsand AD patients' serum samples. Fold change was calculated by 2-Δctmethod. Significant difference among groups were calculated by one-wayANOVA with P<0.05 is considered statistically significant.

FIGS. 3A to 3E show qRT-PCR analysis of AD postmortem brains.Specificity and Expression of (FIG. 3A) miR-455-3p, (FIG. 3B)miR-3613-3p, (FIG. 3C) miR-4674, (FIG. 3D) miR-4668-5p and (FIG. 3E)mir-6722 in AD postmortem brain tissues at Braak Stages (BS) IV, V andVI. Fold change was calculated by 2-ΔΔct method. Significant differenceamong groups were calculated by one-way ANOVA with P<0.05 is consideredstatistically significant.

FIGS. 4A and 4B show the specificity and sensitivity analysis. ROC curveanalysis of miR-455-3p in (FIG. 4A) serum samples from AD patients and(FIG. 4B) AD postmortem brain tissue samples.

FIGS. 5A to 5C show qRT-PCR analysis of miR-455-3p in mice model.mmu-miR-455-3p expression in (FIG. 5A) Cerebral cortex, (FIG. 5B)Cerebellum and (FIG. 5C) Serum of APP-mice compared to wild type mice.Fold change was calculated by 2-ΔΔct method. Significant differenceamong groups were calculated by paired t-test with two-tailed P<0.05 isconsidered significant.

FIGS. 6A and 6B show MiR-455-3p expression in cell lines. qRT-PCRanalysis of hsa-miR-455-3p expression in (FIG. 6A) SHSY-5Y cells, and(FIG. 6B) mmu-miR-455-3p expression in N2a cells. Fold change wascalculated by 2-ΔΔct method. Significant difference among groups werecalculated by paired t-test with two-tailed P<0.05 is consideredsignificant.

FIG. 7 shows a KEGG pathway analysis of miR-455-3p. MiR-455-3p regulatedpathways and target genes were identified using the sources microT-CDSand TarBase to classify KEGG pathway and GO category pathway withP<0.05. MiR-455-3p targeted pathway genes identified through literaturesurvey that were implicated in AD pathogenesis.

FIG. 8 shows MicroRNA 455-3p as a peripheral biomarker for Alzheimer'sdisease.

FIG. 9 shows the details of a microRNA 455-3p study.

FIGS. 10A to 10C show that expression of hsa-miR-455-3p in AD patients.(FIG. 10A) miR-455-3p expression in the postmortem brains of healthycontrols (n=15) and AD patients' (n=32) was quantified by real-timeRT-PCR. Data are presented as “-delta CT” values using box and whiskersplots. Significant difference between groups were calculated by unpairedt-test with P<0.05 is considered statistically significant. (FIG. 10B)Expression of hsa-miR-455-3p in human fibroblast cells from healthycontrols (n=8), Familial AD cases (n=4) and sporadic AD patients' (n=6).Significant difference between groups were calculated by one-way ANOVAwith P<0.05 is considered statistically significant. (FIG. 10C)Expression of hsa-miR-455-3p in human B-lymphocytes cells from healthycontrols (n=10), Familial AD cases (n=6) and sporadic AD patients'(n=6). Significant difference between groups were calculated by one-wayANOVA with P<0.05 is considered statistically significant.

FIGS. 11A to 11C show that ROC curve analysis of hsa-miR-455-3p in (FIG.11A) AD postmortem brains, (FIG. 11B) AD fibroblast cell lines, and in(FIG. 11C) B-lymphocytes cells from AD patients. The curve was plottedbased on the 1CT value of miR-455-3p in AD patients and control samples.Area under the ROC curve (AUROC) was calculated along with thesensitivity and specificity values. P<0.05 is considered statisticallysignificant.

FIGS. 12A to 12D show scattered plot diagrams showing the Pearsoncorrelation coefficient (r) values of miR-455-3p expression with (FIG.12A) AD postmortem brains autolysis time (FIG. 12B) Age of AD postmortembrains (FIG. 12C) Age of AD fibroblast cells and (FIG. 12D) Age of ADB-lymphocytes.

FIGS. 13A to 13H—MiR-455-3p interaction with the APP gene and itseffects on neuroblastoma cells survival (FIG. 13A) The two putativebinding sites of miR-455-3p at the 3′-UTR regions of a wild-type APPgene in humans and mice. Region A (522-528) had 7 nucleotide bindingsites, and Region B (3139-3145) had 6 nucleotide binding sites with theseed sequence of miR-455-3p in both the human and mouse wild-type APPgene (shown in green). (FIG. 13B) Luciferase reporter assay formiR-455-3p binding with the APP gene. Normalized luciferase activity(Firefly/Renilla) in neuroblastoma cells co-transfected with the APP3′UTR clone (HmiT009578-MT06) and miR-455-3p mimics. Firefly/Renillaluciferase activity of the APP 3′UTR clone was significantly decrease bythe miR-455-3p. (FIG. 13C) Representative images of neuroblastoma cellsmorphology after 24 hr of miR-455-3p mimics and inhibitor transfection(10× magnification). Quantitative measurement of miR-455-3p (FIG. 13D),and APP fold-change (FIG. 13E), in cells at 24 hr post mimics andinhibitor transfection. (13F) Representative images of Annexin V and PIstaining of cells at 24 hr post-transfection of miR-455-3p mimics andinhibitor. White arrow represents the viable (green) and dead (red)cells. Percentage (%) of apoptotic cells (FIG. 13G), and viable cellspopulations (FIG. 13H), after 24 hr of miR-455-3p mimics and inhibitortransfection. (*P<0.05) (**P<0.01) (***P<0.001)

FIGS. 14A to 14J—Regulation of mutant APP cDNA expression by miR-455-3p.(FIG. 14A) Representative images for transfection of the miR-455-3pexpression vector (GFP), the miR-control (GFP) vector, and the mutantAPP cDNA in neuroblastoma cells (10× magnification). Bright field imagesand green fluorescence detected in the same field at 24 hrpost-transfection (10× magnification). (FIG. 14B) Representative imagesof Annexin V and Propidium Iodide (PI) stained neuroblastoma cellstransfected with the miR-control vector, the miR-455-3p vector, and themutant APP cDNA. The white arrow indicates the populations of viable(green) cells and dead (red) cells. (FIG. 14C) Percentage (%) of anapoptotic cell population after 24 hr of the miR-455-3p vector, themiR-control vector, and the mutant APP cDNA transfecting cells. (FIG.14D) Percentage (%) of a viable cell population after 24 hr of themiR-control vector, the miR-455-3p vector and the mutant APP cDNAtransfecting cells. (FIG. 14E) Quantitative measurement of miR-455-3pfold change expression after 24 hr of the miR-455-3p vector and themutant APP cDNA transfecting cells. (FIG. 14F) Quantitative measurementof APP mRNA folds change expression after 24 hr of the miR-455-3p vectorand the mutant APP cDNA transfecting cells. (FIG. 14G) Western blot forAPP (6E10), the C-terminal fragment of APP (C99) and B-actin proteins incells transfected with the miR-control vector, the miR-455-3p vector,mutant APP cDNA and co-transfected cells. Quantitative measurement of(FIG. 14H) APP (6E10) and C-terminal fragments of APP C99 (FIG. 14I),and C83 (FIG. 14J) proteins levels by densitometry in the mutant APPcDNA and the miR-455-3p vector transfecting cells. (*P<0.05) (**P<0.01)(***P<0.001).

FIGS. 15A to 15C—MiR-455-3p reduces APP and amyloid proteins. (FIG. 15A)Representative immunostaining images of neuroblastoma cells transfectedwith the miR-control vector, the miR-455-3p vector, the mutant APP cDNA,the mutant APP and miR-455-3p co-transfected. Cells were stain with theAPP (6E10) antibody producing red fluorescence (20× magnification).Green fluorescence showed the miR-control and miR-455-3p vectorexpression and the nucleus of the cell stained with DAPI (blue).Fluorescence intensity (red) of the mutant APP was reduce in mutant APPand miR-455-3p co-transfected cells. (FIG. 15B) Quantitative measurementof fluorescence intensity of the APP (6E10) protein in mutant APP,miR-455-3p, and miR-control vector transfected cells. (FIG. 15C) ELISAanalysis for amyloid ₍₁₋₄₀₎ and ₍₁₋₄₂₎ levels in neuroblastoma cells.Quantitative detection of the (i) human amyloid-β₍₁₋₄₀₎ and (ii)amyloid-β₍₁₋₄₂₎ peptide level in mutant APP cDNA and miR-455-3p vectortransfected cells. (*P<0.05)

FIGS. 16A to 16H—Immunoblotting for mitochondrial biogenesis andsynaptic genes. (FIG. 16A) Representative western blot images for PGC1α,NRF1, NRF2, TFAM, and beta-actin proteins levels in 1) Neuroblastomacells transfected with the miR-control vector, 2) Neuroblastoma cellstransfected with the miR-455-3p expression vector, 3) Neuroblastomacells transfected with the mutant APP cDNA, 4) Neuroblastoma cellsco-transfected with the miR-455-3p and mutant APP cDNA, and 5)Neuroblastoma cells co-transfected with the mutant APP and miR-controlvector. Quantitative measurement of the levels of (FIG. 16B) the PGC1αprotein (FIG. 16C) NRF1, (FIG. 16D) NRF2, and (FIG. 16E) TFAM usingdensitometry in 1) Cells transfected with the miR-control vector, 2)Cells transfected with the miR-455-3p expression vector, 3) Cellstransfected with mutant APP cDNA, 4) Cells co-transfected with themiR-455-3p and mutant APP cDNA, and 5) Cells co-transfected with themutant APP and miR-control vector. (FIG. 16F) Representative westernblot images for synaptophysin, PSD95, and beta-actin proteins levelsin 1) Cells transfected with the miR-control vector, 2) Cellstransfected with the miR-455-3p expression vector, 3) Cells transfectedwith the mutant APP cDNA, 4) Cells co-transfected with the miR-455-3pand mutant APP cDNA, and 5) Cells co-transfected with the mutant APP andmiR-control vector. Quantitative measurement of the levels of (G) thesynaptophysin protein and (FIG. 16H) PSD95 using densitometry in samegroups of cells. (*P<0.05) (**P<0.01)

FIGS. 17A to 17I—Immunoblotting for mitochondrial dynamic genes. (FIG.17A) Representative western blot images for DRP1, FIS1, OPA1, Mfn1, Mfn2and beta-actin proteins levels in: 1) Neuroblastoma cells transfectedwith the miR-control vector, 2) Neuroblastoma cells transfected with themiR-455-3p expression vector, 3) Neuroblastoma cells transfected withthe mutant APP cDNA, 4) Neuroblastoma cells co-transfected with themiR-455-3p and mutant APP cDNA, and 5) Neuroblastoma cellsco-transfected with the mutant APP and miR-control vector. Quantitativemeasurement of the levels of (FIG. 17B) the DRP1, (FIG. 17C) FIS1, (FIG.17D) OPA1, (FIG. 17E) Mfn1 and (FIG. 17F) Mfn2 proteins usingdensitometry in the same groups of cells. (FIG. 17G) Representative TEMimages of Neuroblastoma cells transfected with the miR-control vector,the miR-455-3p vector, mutant APP cDNA, co-transfected with mutant APPand miR-455-3p, and co-transfected with mutant APP and miR-control,showing mitochondrial organization (600 nm magnification). (FIG. 17H)Quantification of the number of mitochondria in the Neuroblastoma cellstransfected with miR-control vector, the miR-455-3p vector, mutant APPcDNA, co-transfected with mutant APP and miR-455-3p, and co-transfectedwith mutant APP and miR-control. (FIG. 17I) Quantification of the sizeof mitochondria (μm) in the neuroblastoma cells transfected withmiR-control vector, the miR-455-3p vector, mutant APP cDNA,co-transfected with mutant APP and miR-455-3p, and co-transfected withmutant APP and miR-control. (*P<0.05) (**P<0.01).

FIG. 18 shows a proposed mechanism of action mt miRNAs on themitochondrial function, ATP production, and synaptic activity.

FIG. 19 is a working model of synaptic miRNAs: miRNAs localized indendrites and axon terminals could modulate the expression of localmRNAs and proteins.

DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not limit the invention, except as outlined in the claims.

Alzheimer's disease (AD) is progressive neurological disorder affectingaged humans (1). The loss of memory, thinking skills, reasoningabilities, and changes in personality and in behavior are the maincharacteristics of AD (2). Currently, over 46.8 million peopleworldwide, including 5.4 million Americans, live with AD-relateddementia, and this number is estimated to increase to 131.5 million by2050 (2). The major pathological hallmarks of AD are the formation ofextracellular amyloid plaques and intracellular neurofibrillary tangles(NFTs) in brains of patients with AD. The amyloid plaques accumulate dueto the overproduction of the amyloid β peptide (Aβ). This overproductionis due to endoproteolysis of the parental amyloid precursor protein(APP), which is cleaved by the enzyme complexes α-, β-, and γ-secretases(3). Increased production and reduced clearance of Aβ in the brain, maylead to a cascade of events in disease process, including synapticdamage, hyperphosphorylated tau (p-tau), mitochondrial structural andfunctional changes, inflammatory responses, hormonal imbalance, cellcycle changes, and neuronal loss (4-6).

Currently, to diagnose AD, several biochemical tests are used to detectAβ and p-tau proteins in the cerebrospinal fluid (CSF) of AD patients.This fluid then undergoes biochemical and molecular tests, to determinethe levels of biomarkers of AD. In the CSF of patients diagnosed withAD, the concentration of Aβ(1-42) has been found to be 40-50% lower thanconcentration levels in individuals who do not have AD (6). Such lowerlevels of Aβ(1-42) in patients have been detected at later stages of ADprogression, but they have not been detected in patients in early stagesof disease progression. The use of CSF analysis to determine levels ofAβ(1-42) is considered a safe procedure, but often times, the patientscomplain about post-examination headaches (7). CSF examination requireshighly skilled persons puncturing the lumbar to remove spinal fluid.Additional testing to diagnose AD uses highly sophisticated neuroimagingtechniques, such as positron emission tomography and structural magneticresonance imaging and scanning (8).

Neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's arevery debilitating diseases. While the cause of Alzheimer's disease isnot known, it is generally accepted that a build up of beta-amyloid (Aβ)plaques in the brain cause the interruption of signaling pathways. Thisbuild up of beta-amyloid (Aβ) plaques causes memory problems, languagedifficulties, mood swings, and other debilitating symptoms. While thereis no cure for Alzheimer's disease, early detection of the disease leadsto a better prognosis.

Example 1

The primary subject matter of the disclosed invention includes the useof MicroRNAs (miRNAs) found in the blood serum to detect the presence orpredict future presence of AD. MicroRNAs are small segments of RNA thatare a recent discovery and are currently being heavily researched.Generally, miRNAs are utilized by the body to regulate certainfunctions. It has been proposed that miRNAs might be useful asbiomarkers for detecting disease where there is no other viable methodof detection.

In the disclosed technology, miRNA-445-3p has been identified as abiomarker for detecting Alzheimer's disease. The results disclosedherein confirm that when a higher level of miRNA-455-3p is found in apatient's serum, this indicates the presence of Alzheimer's with bothhigh sensitivity and specificity. The miRNA levels of known Alzheimer'spatients against non-disease control patients to determine thiscorrelation.

The invention uses miRNA-455-3p as a biomarker for detecting Alzheimer'sat its earliest stage. The only current and definitive test forAlzheimer's is postmortem analysis of the brain. With the methoddisclosed herein, a single blood sample would be capable of measuringthe circulating levels of miRNA-455-3p. These measurements would allowphysicians to more confidently diagnose and detect Alzheimer's in theearliest stages of the disease.

Given these problems with diagnostic tests for AD, in the last decaderesearchers have focused on developing non-invasive diagnostic testscapable of detecting nucleic acids, particularly microRNA (miRNAs),known to regulate in patients with AD. These miRNAs are small nucleotidemolecules (˜22-25 measurement unit) that expressed in humans, plants,fungi, bacteria, and some viruses (9). In neurodegenerative diseaseslike AD, miRNAs have been found to be deregulated in the blood, plasma,serum, CSF, extracellular fluid, and brain tissues of AD patients(10,11).

In humans, miRNAs are believed to be involved in all developmental andpathological processes by regulating gene expression. They achieve thisregulation by targeting 3′ UTR and binding RNA sequences at 3′UTR in asequence-specific manner (12). Some miRNAs are tissue-specific and arelocalized at certain cellular niches, while others are expressed in alltissues and organs of human body. MiRNAs synthesized in the cells andusually modulate mRNA activity of host cells while in severalcircumstances, miRNAs released from cells are involved in regulatingsignals for cells-to-cell communication, known as extracellular miRNAs(13). Extracellular miRNAs are secreted from cells via encapsulatedexosomes and micro-particles, or they are released with severallipoprotein complexes, such as high-density lipoproteins (HDL),low-density lipoproteins (LDL), and argonaute 2 proteins (13). Theseextracellular circulatory miRNAs are very stable in blood components. Inpathological conditions, such as in persons with AD, concentrations ofparticular miRNAs are altered (11). However, the inventors still do nothave complete understanding of how expressions(s) of miRNAs progress innon-demented elderly individuals to mild cognitive impairment (MCI), andMCI to AD.

Several recent miRNA studies using CSF, serum, plasma and whole bloodrevealed that circulatory miRNAs as peripheral biomarkers in AD(6,14-22). However, these studies provided information about miRNAs withlittle or no consensus in all studies. Further, validation ofdifferentially expressed miRNAs using AD postmortem brains is not welldone in these studies.

Therefore, a more detailed study on circulatory miRNAs in AD patientsand MCI subjects with thorough validation is urgently needed, in orderto determine early detectable peripheral biomarkers in AD. In thepresent study, the inventors screened AD patients, MCI subjects, andhealthy controls for circulatory miRNAs in serum samples using anAffymetrix microarray and qRT-PCR validation assay. Further,differentially expressed miRNAs were validated using AD postmortembrains, APP transgenic mice and AD cell lines.

Levels of serum miRNAs. Total RNA was extracted from 40 serum samples,for microarray analysis, the concentration of miRNAs (10-40 nucleotides)and small RNAs (0-257 nucleotides), and the ratio of miRNAs to smallRNAs in each sample were analyzed. The RNA levels were calculated by anAgilent 2100 Bioanalyzer (Agilent Technologies). The averageconcentration of miRNAs in AD patients was 89.1 pg/μl, in MCI subjects,132.7 pg/μl and in controls was 119.3 pg/μl. The average concentrationof small RNAs was 186 pg/μl in AD patients, 248.5 pg/μl in MCI subjectsand 240.2 pg/μl in controls. Similarly, the ratios of average miRNAs tosmall RNAs in the samples were 49.1%, 54.9% and 49.8% in AD patients,MCI subjects and in controls respectively. These results indicated thatmiRNA output was greater in the MCI subjects.

Primary screening of serum samples to detect miRNAs. AD patients (n=10),MCI subjects (n=16), and controls (n=14) were analyzed for their miRNAmicroarray expression using the Affymetrix GeneChip miRNA Array, v. 4.0.A total of 6631 genes were detected in all of the serum samples. Ofthese 6631 genes, 2578 were mature miRNAs that were listed in themiRbase database, and 2025 were the stem-loop precursor miRNAs(pre-miRNAs). The remaining genes belonged to different classes of smallRNAs, such as snoRNA (1491), CDBox (319), HAcaBox (155), scaRna (31),and 5.8s rRNA (10). Of the remaining genes, 22 were spike-in controlRNAs that were added externally during the array experiment.Differential miRNA expression in each miRNA was analyzed, on thefold-change intensity of each miRNA (−2 to +2) and each ANOVA P-value(<0.05).

AD patients and healthy controls. Microarray analysis was performed onthe samples from the AD patients (n=10) and the controls (n=14) (FIG.1). The miRNA bi-weight average (log 2) intensity showed significant(P<0.05) deregulation of 7 miRNAs in AD patients compared to controls(Table 1). The miRNA sequences, hsa-miR-455-3p, hsa-miR-3613-3p,hsa-miR-4668-5p, hsa-miR-5001-5p, hsa-miR-4674, and hsa-miR-4741 wereup-regulated, while hsa-miR-122-5p was down-regulated. The top miRNAcandidate was hsa-miR-455-3p, which showed a remarkably 11.3-fold higherexpression in AD patients compared to controls. Other miRNAs were,hsa-miR-3613-3p (3.67-fold), hsa-miR-4668-5p (3.38-fold), andhsa-miR-4674 (5.62-fold) also exhibited the higher levels of foldexpression in AD patients. These results identified new miRNA candidatesthat were not previously identified in AD.

TABLE 1 MiRNAs: 1og2 intensity and fold change in AD patients andcontrols Fold AD Bi-weight AD Control Bi-weight Control ChangeTranscript miRNA Average Signal Standard Average Signal Standard(linear) ANOVA FDR Cluster ID name (log2) Deviation (log2) Deviation (ADvs. Control) p-value p-value Chromosome 20504187 hsa- 6.03 1.05 2.531.06 11.3 0.000003 0.007 chr9 miR- 455-3p 20517821 hsa- 3 1.32 1.13 0.163.67 0.000014 0.012 chr13 miR- 3613-3p 20519463 hsa- 3 1.5 1.25 0.423.38 0.000576 0.079 chr9 miR- 4668-5p 20500726 hsa- 2.31 1.4 4.26 1.24−3.85 0.004833 0.198 chr18 miR- 122-5p 20520198 hsa- 3.73 0.7 2.49 0.772.37 0.011848 0.263 chr2 miR- 5001-5p 20519474 hsa- 4.48 1.37 1.99 1.055.62 0.013827 0.275 chr9 miR- 4674 20519592 hsa- 2.31 0.61 1.26 0.422.07 0.013923 0.276 chr18 miR- 4741

AD patients and MCI subjects. To compare the intermediate states ofdisease progression, microarray data were analyzed from the AD patients(n=10) and MCI subjects (n=16). Heat map data and hierarchicalclustering showed the differential expressions of 8 miRNA candidates.Based on the bi-weight average (log 2) intensity and linear fold-changevalues, the miRNAs hsa-miR-3613-3p and hsa-miR-4668-5p weresignificantly up-regulated (ANOVA, P<0.05) and hsa-mir-320d-2,hsa-miR-378h, hsa-miR-3921, hsa-miR-6805-5p, hsa-miR-92a-3p,hsa-miR-3613-5p were down-regulated in the serum samples of AD patientscompared to MCI subjects.

MCI subjects and healthy controls. Microarray data were compared betweenMCI subjects (n=16) and controls (n=14). Hierarchical clustering showeda wide range of miRNA signatures that were deregulated. Interestingly,50 miRNAs were identified, and all of them were significantlyup-regulated in MCI subjects. Surprisingly, miR-4674 (5.24-fold) andmiR-455-3p (5.18-fold) showed maximum upregulation in MCI subjects,compared to controls. These results suggested that greater number ofmiRNA deregulation were observed in the initial phases of diseaseprogression.

AD patients, MCI subjects, and healthy controls. To detect diseaseprogression through the differential expression of miRNAs, the inventorscompared the differentially expressed miRNAs in the serum samples of ADpatients and MCI subjects (n=10 and n=16, respectively) and controls(n=14) at the same time point in disease progression. The miRNAs in eachgroup of serum samples were analyzed in terms of: bi-weight average (log2) intensity, a fold change of less than −2 or more than 2, and anANOVA/FDR, P<0.05. Results indicated that a total of 68 miRNAs (32mature and 36 precursor) were deregulated among three groups of serumsamples. Since, present study aimed to identify promising biomarkers forAD progression, the inventors focused on miRNAs, and those expressionsare either gradually increased or decreased among three groups. Of the32 mature miRNAs that were identified, 7 were gradually upregulated:hsa-miR-455-3p, hsa-miR-3613-3p, hsa-miR-4674, hsa-miR-4668-5p,hsa-miR-4317, hsa-miR-3124-3p, and hsa-miR-6856-3p, while one,hsa-miR-1972 was down-regulated in AD and MCI subjects compared tocontrols. Further, the remaining pre-miRNAs (hsa-mir-124-1,hsa-mir-4417, hsa-mir-1908, hsa-mir-3912, hsa-mir-4325, and hsa-4776-2)showed gradual upregulation while 4 of the pre-miRNAs (hsa-mir-6722,hsa-mir-412, hsa-mir-3153, and hsa-mir-4430) showed gradualdownregulation.

Among the 68 miRNAs that the inventors studied, the most significantlyupregulated (ANOVA/FDR, P<0.05) were 4 miRNAs miR-455-3p, miR-3613-3p,miR-4674, and miR-4668-5p and the one down-regulated miRNA mir-6722.These miRNAs were selected for secondary screening and validationanalysis since their expression varied more in the 3 groups of serumsamples (Table 2). The miR-455-3p log 2 intensity showed a 2.53-foldincrease in the controls, a 4.9-fold increase in MCI subjects, and a6.03-fold increase in AD patients. Similarly, the expression of miR-4674also increased from 1.99-fold in controls, to a 4.38-fold increase inMCI subjects, and to a 4.48-fold increase in AD patients.

TABLE 2 miRNAs: 1og2 intensity and fold change in AD patients, MCIsubjects, and controls Controls AD Bi- MCI Bi- Bi- Fold Fold Fold weightweight weight ANOVA FDR Change Change Change Average Average Averagep-value p-value (linear) (linear) (linear) Transcript Transcript SignalSignal Signal Controls (All (All (AD vs. (AD vs. (MCI vs. Cluster ID ID(log2) (log2) (log2) ADSD MCISD SD Conditions) Conditions) MCI)Controls) AD) 20504187 hsa- 6.03 4.9 2.53 1.05 1.29 1.06 0.0000020.001994 2.18 11.3 −2.18 miR- 455-3p 20537464 hsa- 5.72 6.27 6.72 0.450.48 0.4 0.000016 0.007169 −1.46 −1.99 1.46 mir- 6722 20519474 hsa- 4.484.38 1.99 1.37 1.26 1.05 0.000208 0.024285 1.07 5.62 −1.07 miR- 467420517821 hsa- 3 1.25 1.13 1.32 0.64 0.16 9.42E-07 0.001562 3.37 3.67−3.37 miR- 3613-3p 20519463 hsa- 3 1.67 1.25 1.5 0.91 0.42 0.0007110.045154 2.52 3.38 −2.52 miR- 4668-5p

The miR-4668-5p showed a gradual upregulation of up to 3-fold in ADpatients, compared to 1.25-fold increase in controls, and a 1.67-foldincrease in MCI subjects. The miR-3613-3p expression also graduallyincreased when the miRNAs were analyzed and compared in controls, MCI,and AD patients (1.13-fold, 1.25-fold, and 3.0-fold increases,respectively). The expression of mir-6722 gradually decreased in MCIsubjects (6.27-fold) and AD patients (5.72-fold) compared to mir-6722expression (6.72-fold) in controls. Thus, the circulatory serum miRNAsshowed aberrant expression in the healthy controls and diseased states(AD and MCI). Also noteworthy was that the level of expression in thesemolecules consistently either increased or decreased with diseaseprogression. Hence, such miRNAs are capable of discriminating betweenhealthy persons and persons with AD or MCI. Such miRNAs could also beused to monitor disease progression in AD patients.

Secondary screening and validation of miRNAs in serum samples. SelectedmiRNAs (miR-455-3p, miR-3613-3p, miR-4674, miR-4668-5p and mir-6722)were further validated for their expression using qRT-PCR assays. Theexpression of miR-455-3p was quantified in serum of controls (n=18), MCIsubjects (n=20), and AD patients (n=11). Interestingly, fold-change(mean±SD) analysis indicated a gradual upregulation of miR-455-3p in ADpatients (0.071±0.078-fold) (P=0.007) compared to the fold-change in MCIsubjects (0.034±0.024-fold) and in controls (0.019±0.020-fold) (FIG.2A). Similarly, the expression of miR-4668-5p was also significantly(P=0.016), upregulated in MCI subjects (2.25±2.78-fold) compared tocontrols (0.340±0.50-fold) (FIG. 2B). However, miR-4668-5p expressiondid not show significant elevation in the AD patient's serum(1.50±1.61-fold). In similar way, expressions of miR-4674 andmiR-3613-3p also increased in the MCI and AD patients, though it was notsignificant (FIGS. 2C and 2D).

The expression of precursor miRNA (mir-6722) was also quantified byqRT-PCR, with results showing a gradual down regulation in MCI and ADpatients compared to controls, but not significantly (FIG. 2E). Amicroarray-based panel of 5 miRNAs was found to concur with qRT-PCRvalidation in controls, MCI and AD serum samples. However, a statisticalanalysis revealed that miR-455-3p and miR-4668-4p were significantlyupregulated in persons with AD or MCI from healthy controls.

Validation of serum miRNA expressions using AD postmortem brains. TotalRNA was isolated from postmortem brains (frontal cortex) of AD patientsat Braak stages IV (n=4), V (n=6), and VI (n=6), and in controls (n=5).Expression of selected 5-miRNA panel was quantified by qRT-PCR. Theaverage fold change in each miRNA was higher in AD brains at Braakstages IV, V, and VI compared to the control brains. Expression ofmiR-455-3p was increased in the AD brains at all Braak stages comparedto controls. However, a significant upregulation was observed in brainsfrom AD patients at the Braak stage V (26.59-fold, P=0.016) (FIG. 3A).Similarly, miR-3613-3p expression was also higher in the brain tissuesat Braak stage V (P=0.003) compared to controls (FIG. 3B).

MiR-4674 expression was also higher in the postmortem brains from ADpatients at the Braak stages IV and V, but it was significantly higherat stage VI (P=0.003) (FIG. 3C). MiR-4668-5p expression was alsoup-regulated in AD brains at Braak stages IV, V, and VI, but not thesignificant level (FIG. 3D). Mir-6722 expression was down-regulated inAD serum samples. However, the expression of mir-6722 was surprisinglyincreased in the AD patients at Braak stage VI (P=0.018) (FIG. 3E). Theopposite facts were observed in the analysis of mir-6722 expression.

The upregulation of miR-455-3p was significant in postmortem brains fromAD patients at Braak stage V. Interestingly, those individuals were allhaving the ApoE (3/4) genotype. The expression of miR-455-3p was themost significantly higher in both the AD serum and AD postmortem brainssuggesting that it might be implicated in AD detection and pathogenesis.

Receiver operating characteristics (ROC) curve analysis of miR-455-3p.To determine the diagnostic accuracy using miRNAs in AD patients, ROCcurves analysis was studied for miR-455-3p expressions in serum and ADbrain samples. The curves were plotted, based on the ΔCt value ofmiR-455-3p expression in serum samples from the AD patients (n=1) andcontrols (n=18). Upon analysis, miR-455-3p showed significant area undercurve (AUC). The AUROC=0.79 with a 95% confidence interval was 0.59 to0.98 (P=0.015) in AD serum samples compared to the healthy controls(FIG. 4A). Further, ROC analysis of miR-455-3p expression in postmortembrains from 16 AD patients and 5 healthy controls indicated thesignificant AUROC value of 0.86 (95% confidence interval was 0.61 to1.11, P=0.016) (FIG. 4B). Thus, ROC analysis confirmed miR-455-3p as avaluable molecule capable of discriminating persons with and without AD.

Expression of miR-455-3p in APP transgenic mice. Since miR-455-3p showedpromising results in terms of its expression in AD serum samples and ADpostmortem brains, miR-455-3p expression was also studied in thecortical tissues from an APP transgenic mouse model of AD (Tg2576 line).This study investigated the mmu-miR-455-3p expression in brain tissuesfrom 6-month-old APP mice (n=6) and C57BL/6 wild-type mice (n=6). TotalRNA was extracted from disease-affected tissue from the cerebral cortexand non-affected-cerebellum, and mmu-miR-455-3p expression was measuredby qRT-PCR. Results showed a 1.8-fold (P=0.004) upregulation ofmmu-miR-455-3p in the cerebral cortex tissues of the APP mice, comparedto the wild type mice (FIG. 5A). Interestingly, in cerebellum,mmu-miR-455-3p expression was significantly (P=0.018) reduced in the APPmice (FIG. 5B). Expression of mmu-miR-455-3p was also examined in theserum samples from APP mice. Mmu-miR-455-3p expression was higher in theAPP mice serum compared to the wild-type mice, although it was notstatistically significant (FIG. 5C). A high level of mmu-miR-455-3pexpression in the APP mice confirmed a possible role in Aβ-mediated ADpathogenesis.

MiR-455-3p expression in Aβ(1-42) treated cells. To determine theeffects of the Aβ on the expression of miR-455-3p, the SH-SYSY (humanneuroblastoma) and N2a (mouse neuroblastoma) cells were treated with theAβ(1-42) peptide (20 μM) for 6 hours. Total intracellular RNA wasextracted and expression of human and mouse miR-455-3p were measured byqRT-PCR. Results showed a 4.1-fold (P=0.027) increase in hsa-miR-455-3pexpression in the Aβ-treated SH-SYSY cells compared to control(untreated) cells (FIG. 6A). Similarly, in N2a cells, mmu-miR-455-3pexpression was also upregulated by 3.8-fold (P=0.021) in Aβ-treatedcells compared to control cells (FIG. 6B). These results furtherconfirmed the significant response of miR-455-3p in Aβ pathologies.

MiRNA-associated signaling pathways. MiRNA-associated signaling pathwayswere analyzed using DIANA TOOLS-miRPath algorithm to identify thebiological function of these miRNAs and their role in AD pathogenesis.MicroT-CDS files for all five miRNAs were run with a p value <0.05. KEGGpathway analysis unveiled more than 54 biological pathways associatedwith these miRNAs. This analysis focused on the miR-455-3p andidentified possible molecular targets involved in AD pathogenesis. ThemiRNA analysis identified the relationship of miR-455-3p with 11biological pathways and associated genes (FIG. 7). The most importantsignaling pathways were: the ECM-receptor interaction, the adherensjunction, the TGF-beta signaling pathway, the hippo signaling pathway,and the regulation of the actin cytoskeleton. These signaling pathwaysand some of their genes (THBS1, COL3A1, HSPG2, COL6A1, RUXN1, MYC,Smad2, PLK1, and TNC) were directly associated with AD pathogenesis. Theupregulation of miR-455-3p in AD development might be associated withthe modulation of the above-mentioned genes. Thus, analysis of thesignaling pathways revealed a possible molecular mechanism for howmiR-455-3p is involved in AD pathogenesis.

Despite the enormous research efforts that have gone into developingways to diagnose AD at the earliest stages possible, little progress hasbeen made. Perhaps this is at least in partly due to initial researchthat focused on personal characteristics of persons who developed AD,such as their life style, body mass index, status of AD-related alleles,their genotypic and phenotypic variations, and environmental factors(11,23,24). This research recently broadened the list of potentialbiomarkers for AD to include blood-based miRNAs, but until the last 5-8years, little research was actually conducted to narrow the list ofmiRNAs that might serve as biomarkers for AD, since more than onehundred miRNAs were found to be deregulated in AD patients (6,11,14-21).

The present study narrows the field of miRNAs that might serve asperipheral biomarkers for AD. Using Affymetrix microarray analysis, theinventors identified about 6631 types of small RNAs in the serum ofpatients with AD and with MCI. Of these, only 2578 were mature, and 2025were stem-loop precursors of human miRNAs. These numbers of maturemiRNAs were almost same as the miRNAs entry on miRbase release 21 (2588mature) for Homo sapiens (www.mirbase.org/cgi-bin/browse.pl). From theseresults, the inventors came to know that most of genomic miRNAs werepresent and/or secreted in peripheral circulation. Based on a recentliterature of circulatory miRNAs (11), the inventors had expected thatdisease-specific miRNAs or miRNAs associated with a particularpathological state, such as AD, were differently expressed and releasedin peripheral circulation (11). When the inventors compared the 3different study groups in this study (serum from AD patients, MCIsubjects and healthy controls), it was found that miRNA expressions of awide number of miRNAs change, depending on the serum-donor's stage ofdisease progression. The highest variation of miRNA expressions wasfound in the patients' serum who were at the initial stage of diseaseprogression, when the diagnosis of these patients went from control toMCI. Hence, disease-specific early physiological changes are crucial forthe miRNAs deregulation in cells. Sequencing analysis of serum exosomesunveiled differential expression of 17 miRNAs in the serum from 3subject groups (17). However, due to low number of MCI subjects (n=11),none of the exosomal miRNA was verified as biomarker for diseaseprogression (17). In present study, five miRNAs (miR-455-3p,miR-4668-5p, miR-3613-3p, miR-4674, and mir-6722) were selected forvalidation in order to determine potential biomarker. Results showed aremarkable variation of miR-455-3p in AD serum samples, AD postmortembrains and AD mice, suggesting that it is potential biomarker for AD.These five miRNAs are known to have specific regulating roles indifferent diseases. MiR-455-3p has a role in colon cancer (25) and alsoparticipates in chondrogenic differentiation (26) and cartilagedevelopment and degeneration (27). In patients with preeclampsia,miR-455-3p was also found to be linked with hypoxia signaling and theregulation of brown adipogenesis via the HIF1an-AMPK-PGC1α signalingnetwork (28,29). In familial amyotrophic lateral sclerosis,downregulation of miR-455-3p expression has been reported in the sera ofthese patients (30). The roles of miR-3613-3p and miR-4668-5p have beenstudied in the pathogenesis and progression of IgA nephropathy (31) andin mesial temporal lobe epilepsy (32). In the plasma of AD patients, lowlevels of miR-3613-3p were detected using RNA sequencing (33). However,current study showed increased levels of miR-3613-3p in the serumsamples of AD patients. In hemolysis-free blood plasma from prostaticcancer patients, an increased level of miR-4674 was reported (34).However, role of these miRNAs is not widely reported in AD and otherneurodegenerative diseases.

A recent analysis of biofluids (serum, plasma, CSF) from AD patientsrevealed many miRNA potential biomarkers for AD, such as miR-9,miR-125b, miR-146a, miR-181c, let-7g-5p, and miR-191-5p (11). However,their expression levels and molecular characterizations were notinvestigated using postmortem brains from AD patients and AD cell andmouse models. Consequently, no miRNA has been identified as the mostlikely biomarker for AD. In this current study, the inventors analyzedsera and cortices from AD patients found a significant upregulation ofmiR-455-3p. The inventors attempted to replicate these observations inAD postmortem brains, APP transgenic mice, and AD cell lines.Interestingly, miR-455-3p expression was more significantly up-regulatedin the brains and sera from AD patients at Braak stage who had the ApoE(3/4) genotype. These observations unveiled a possible molecularinteraction between miR-455-3p and the ApoE4 genotype.

Another significant finding was that APP transgenic mice exhibited Aβpathologies (35,36) that corresponded to their high expression ofmiR-455-3p in the disease-affected brain cortex, but not in other brainareas known not to be affected by disease, such as the cerebellum. Thesefindings indicate a possible molecular link between APP processing andmiR-455-3p. This hypothesis was further tested on human and mouseneuroblastoma cells treated with toxic Aβ(1-42) peptides which mimicsthe AD type pathophysiology. Higher expression levels of miR-455-3p inthe Aβ(1-42)-treated cells further support miR-455-3p as a potentialbiomarker for AD. This study is the first to identify miR-455-3p as akey molecule expressing biomarker properties for AD.

MiR-455-3p expression is regulated by the transforming growth factorbeta (TGF-β) (37), and its level of expression has been found to beinduced by TGF-β1, TGF-β3, and activin A in human SW-1353 chondrosarcomacells and murine C3H10T1/2 cells (28). The TGF-β signaling pathwayreported to play a critical role in Aβ processing in patients with ADsince reduced TGF-β signaling has been found to be increased in Aβdeposits in patients with AD (38,39).

Through pathway analysis, the KEGG pathway was found to regulate theTGF-β signaling pathway and eleven associated genes by miR-455-3p (FIG.7). As a consequence, miR-455-3p is interconnected with TGF-β signalingand Aβ synthesis, and hence, may play a crucial role in AD pathogenesis.MiR-455-3p might also have a major regulatory role in other cellularpathways through modulation of their genes in AD pathogenesis (FIG. 7).It is possible that miR-455-3p may be involved in AD progression throughaltered expressions of HSPG2, THBS1, COL3A1, COL6A1, TNC, MYC, Smad2,RAN, PLK1, TP73, ACTN1, and IQGAP1 genes (39-47).

FIG. 8 shows MicroRNA 455-3p as a potential peripheral biomarker forAlzheimer's disease. To identify the early peripheral microRNA (miRNA)biomarkers for Alzheimer's disease (AD), the present inventors useddifferent sources of materials in this study. Serum samples andpostmortem brains from AD patients, brain tissues from AD transgenicmice and AD cell lines were used. Primary screening of serum samplesfrom AD patients, MCI individuals and healthy subjects showedderegulated expression of five miRNAs by global Affymetrix basedmicroarray analysis. MiRNAs were further validated in serum samples formAD patients, MCI individuals and healthy controls. Out of five, onlymiR-455-3p showed the most significant upregulation in AD and MCI casescompared to controls. Postmortem brains form AD patients also showed thesignificant upregulation of miR-455-3p in AD cases compared to controls.miR-455-3p expression also upregulated in the frontal cortices of APPtransgenic mice compared to wild type mice. Finally, the inventorstested the miR-455-3p expression in human and mouse neuroblastoma celllines treated with Amyloid-beta 42 peptide. In Amyloid-beta 42 treatedcells, miR-455-3p expression was also elevated compared to controlcells.

FIG. 9 shows the details of a microRNA 455-3p study. Lymphoblasts andfibroblasts from AD patients and healthy controls (obtained from NIAcell line repositories) can be used in order to establish miR-455-3p asperipheral biomarker for Alzheimer's disease. Expression of miR-455-3pcan be correlated with the levels of AD proteins. The molecularproperties of miR-455-3p can also be used to mimic (overexpression) and(inhibitor) knockout approaches in human and mouse neuronal cells. Theseexperiments can be used to determine the deleterious and protectiveeffects of miR-455-3p in AD neurons. Using overexpression and/orknockouts of miR455-3p in APP transgenic and knockout mice it ispossible to study the molecular features of miR455-3p during diseaseprogression of AD. Finally, miR-455-3p transgenic and miR-455-3pknockout mice lines can be generated and used to explore the role ofmiR-455-3p in maintaining synaptic, mitochondrial and inflammatoryfunctions and cognitive behavior.

In summary, the present inventors have identified multipledifferentially expressed circulatory miRNAs in AD patients and subjectswith MCI relative to healthy controls. A careful validation ofdifferentially expressed miRNAs using AD postmortem brains, APPtransgenic mice and AD cell lines revealed that miR-455-3p could be apotential diagnostic biomarker for AD. Further research is still neededto better understand the role of miR-455-3p in AD progression andpathogenesis.

Study subjects. Sera and DNA samples were collected from patients underthe FRONTIERS project based at Garrison Institute on Aging (GIA), TexasTech University Health Sciences Center (TTUHSC). These samples wereobtained from 11 patients diagnosed with AD, 20 patients with MCI, and18 healthy controls. The study protocol was approved by theInstitutional Review Board for FRONTIERS, and all subjects providedinformed written consent. All the bio-specimens were stored at GIABio-Bank. Patient information on demographic characteristics, medicalhistory, biochemical profiles, and their risk factors for AD wasgathered with a standardized questionnaire. Demographic and clinicalcharacteristics of subjects are listed in the Table 3. After completingthe questionnaire, all subjects underwent a detailed clinicalexamination to evaluate them for inclusion and exclusion criteriaestablished by NINCDS-ADRDA. The inclusion criteria were: (1) 45 yearsand above age, (2) rural community based West Texas individuals, and (3)all study participants have assessed for cognitive functions. Theexclusion criteria were: (1) individuals with strong medication and (2)too many health complications.

MiRNAs extraction. MiRNAs, including other small RNAs, were extractedfrom the serum samples with the miReasy serum/plasma kit (Qiagen,Germany) as per manufacturer's instructions (16). Briefly, 200 μl ofserum samples were mixed with 5 volumes of a Qiazol lysis reagent and anequal volume of chloroform, and centrifuged to separate the aqueousphase. MiRNAs accumulated in the aqueous phase were precipitated with100% ethanol. MiRNAs were washed with a buffer and 80% ethanol, andpurified miRNAs were eluted in 15 μl of RNase-free water. RNA qualityand quantity were measured by NanoDrop2000c (Thermo Scientific, USA).

Primary miRNAs screening by Affymetrix microarray. Detailed miRNAsscreening of the serum samples were conducted in the University of TexasSouthwestern Medical Center, Genomics and Microarray Core Facility,Dallas. The miRNA expression profiles were generated with AffymetrixGeneChip miRNA array v. 4.0 (Affymetrix). The GeneChip miRNA 2.0 arrayscontain a 100% miRBase version 20 coverage: 30,424 mature miRNAs werefrom all organism; 5,214 from human, rat, and mouse miRNAs; and 1,996from human snoRNA and scaRNA. It also provided 3,770 probe sets that areunique to human, mouse, and rat pre-miRNA hairpin sequences. TheGeneChip miRNA 4.0 array demonstrated superior performance with 0.95reproducibility (inter- and intra-lot) and >80% of transcripts weredetected at 1.3 amol from 130 ng of total RNA. Data were represented bythe GeneChip miRNA 4.0 array in 4 logs that correlated with a dynamicrange of >0.97 signal and >0.94 fold-change.

Briefly, 8 μl of total RNA was treated for poly (A) tailing reaction at37° C. for 15 min as per the protocol. 4 μl of 5× Flash Tag Biotin HSRligation mix was added to poly (A) tailed RNA, and the mixture wasincubated at 25° C. for 30 min, using the Flash Tag Biotin HSR Labelingkit following manufacturer's instructions (cat. no. HSR30FTA;Genisphere, LLC, Hatfield, Pa., USA). Biotin HSR that labeled with RNAwas mixed with an array hybridization cocktail according to the GeneChipEukaryotic Hybridization control kit manual and was processed using theAffymetrix GeneChip miRNA array. Samples were incubated on thehybridization array chip at 48° C. and 60 rpm for 16 to 18 hours. Afterhybridization, the chips were washed and stained by GeneChiphybridization, washed again, and then stained with an Affymetrix kitaccording to the manufacturer's protocols. The hybridized chips werescanned with an Affymetrix GCS 3000 7G Scanner (48).

Microarray data analysis. Raw data were obtained, using the AffymetrixGeneChip array in the form of an individual CHP file. Each sample wasthen analyzed, using Transcriptome Analysis Console software v. 3.Tukey's bi-weight average (log 2) intensity was analyzed with an ANOVAp-value (<0.05) and FDR p-value (<0.05) for all conditions, for allgenes in the samples from AD, MCI and control group. SAM (significanceanalysis of microarray) with the R package was used to identifydifferentially expressed miRNA and gene probe sets in samples from theAD patients and the controls. Probe sets were considered biologicallysignificant if the fold changes were 2 (49).

Validation of serum miRNAs expression using qRT-PCR. (i)Polyadenylation—One g of total RNA was polyadenylated with an miRNAFirst-Strand cDNA synthesis kit (Agilent Technologies Inc., CA, USA),following manufacturer's instructions. Briefly, a polyA reaction wasprepared by mixing RNA with 4.0 μl of 5× poly A polymerase buffer, 1.0μl of rATP (10 mM), 1 μl of E. coli poly A polymerase, producing a finalvolume of 20 μl with RNase-free water. The tube with these componentswas incubated at 37° C. for 30 min, followed another incubation at 95°C. for 5 min to terminate the adenylation reaction (50). (ii) cDNAsynthesis—Ten μl of polyadenylated miRNAs were processed for cDNAsynthesis with the miRNA First-Strand cDNA synthesis kit (AgilentTechnologies Inc.). The following reaction components were combined in atube: 2 μl of 10× AffinityScript RT buffer, 0.8 μl of dNTP mix (100 mM),1 μl of RT adaptor primer (10 μM), 1.0 μl of AffinityScript RT/RNaseBlock enzyme, and polyadenylated RNA. The combination resulted in areaction volume of 20 μl RNase-free water. This reaction mixture wasincubated at 55° C. for 5 min, then at 25° C. for 15 min, followed by anincubation at 42° C. for 30 min, and a final incubation at 95° C. for 5min in a Veriti 96 well thermal cycler (Applied Biosystems, USA).Resulting cDNAs were diluted with 20 μl of RNase-free water and storedat 800 C for further analysis. (iii) qRT-PCR for miRNAs—qRT-PCR reactionwas performed by preparing a reaction mixture containing 1 μl ofmiRNA-specific forward primer (10 μm), 1 μl of a universal reverseprimer (3.125 μm) (Agilent Technologies Inc., CA, USA), 10 μl of 2×SYBR®Green PCR master mix (Applied Biosystems, NY, USA), and 1 μl of cDNA. Tothis mixture RNase-free water was added up to a 20 μl final volume.Primers for hsa-miR-455-3p, miR-4674, miR-3613-3p, miR-4668-5p, andmir-6722 were synthesized commercially (Integrated DNA Technologies,Inc. Iowa USA) (Table 3).

TABLE 3 miRNAs primers sequences for qRT-PCR SEQ ID Base NO: miRNASequence (5′ to 3′) pairs 1 Hsa-miR-455-3p F-GCAGTCCATGGGCATATACAC 21 2Mmu-miR-455-3p F-GCAGTCCACGGGCATATACAC 21 3 Hsa-miR-4674-3pF-CTGGGCTCGGGACGCGCGGCT 21 4 Hsa-miR-4668-5p F-AGGGAAAAAAAAAAGGATTTGTC23 5 Hsa-miR-3613-3p F-ACAAAAAAAAAAGCCCAACCCTTC 24 6 Hsa-mir-6722F- GGCCTCAGGCAGGCGCACCCGA 22 7 R- GGGTGGGCCAGGCTGTGGGGCG 22 8 U6 snRNAF- CGCTTCGGCAGCACATATACTAA 23 9 R- TATGGAACGCTTCACGAATTTGC 23 10snoRNA-202 F-AGTACTTTTGAACCCTTTTCCA 22

To normalize the miRNA expression, U6 snRNA (small nuclear RNA)expression was also quantified in the serum samples, which was used asan internal control. The reaction mixture of each sample was prepared intriplicates. The reaction was set in the 7900HT Fast Real Time PCRSystem (Applied Biosystems, USA) using following reaction conditions:initial denaturation at 95° C. for 5 min, denaturation at 95° C. for 10sec, annealing at 60° C. for 15 sec, and extension at 72° C. for 25 sec.The relative levels of miRNAs in the AD patients versus the controls andversus the MCI subjects were determined in terms of their fold change,using the formula (2^(−ΔΔct)), where ΔCt was calculated by subtractingCt of U6snRNA from the Ct of particular miRNAs, and ΔΔCt value wasobtained by subtracting ΔCt of particular miRNAs in the controls fromthe ΔCt of miRNAs in the AD and MCI. qRT-PCR was performed intriplicate, and the data were expressed as the mean±SD (50,51).

Postmortem brains from AD patients. Postmortem brain tissues wereobtained from the GIA Brain Bank. The frontal cortices of the postmortembrains were dissected from the AD patients (n=16) and controls (n=5).Demographic details of study participants were given in Table 4. Thestudy protocol was approved by the Institute Ethical Committee atTTUHSC, and brain tissue was obtained after written informed consentfrom the deceased's relatives.

TABLE 4 Characteristics of postmortem brain tissues from controls and ADpatients Cases Gender Age (yrs) Braak Stage PMI (hrs) ApoE HC1 M 71 — 93/4 HC2 M 68 — 6.5 3/4 HC3 F 72 — 11.5 3/3 HC4 F 71 — 7.5 2/2 HC5 M 82 —6.25 3/3 AD1 M 82 IV — 3/4 AD2 M 62 IV 5 3/3 AD3 M 78 IV 7.5 3/4 AD4 M91 IV 8 3/3 AD5 F 77 V 4 3/4 AD6 F 86 V 3.5 3/4 AD7 F 86 V 5 3/4 AD8 F75 V 4 3/4 AD9 F 80 V 5 3/4 AD10 M 78 V 7 3/4 AD11 M 74 VI 7 3/4 AD12 F81 VI 6.25 3/3 AD13 F 83 VI 9.25 3/4 AD14 F 86 VI 6 3/4 AD15 M 84 VI 83/3 AD16 M 82 VI 5.25 3/4

MiRNAs extraction and qRT-PCR. MiRNAs extraction and cDNA synthesis werefollowed as described above, while total RNA was isolated from the 80 mgof frontal cortices using the TriZol RT reagent (Ambion, USA) as permanufacturer instructions. Briefly, tissue samples were homogenized in 1ml of TriZol reagent with Bio-Gen PRO200 Homogenizer (PRO ScientificInc., CT, USA) in a 2-ml RNase-free tube. Chloroform (0.2 ml) was addedto the tissue homogenate, vigorously shaken for 15 seconds, and storedfor 5 min at room temperature. The mixture was then centrifuged at12,000 g for 15 min at 4° C. The supernatant was transferred to a newtube and precipitated with 0.5 ml of isopropanol for 15 min at roomtemperature. Samples were centrifuged at 12,000 g for 10 min at 4° C.The resulting RNA pellet was washed with 1 ml of 75% ethanol andcentrifuged at 7,500 g for 5 min at 4° C. The RNA pellet was dried anddissolved in 20 μl of DEPC-treated water. The quality and quantity ofthe RNA were analyzed by NanoDrop analysis. The value of absorbance ofeach brain RNA sample (A₂₆₀/A₂₈₀) was 1.8 to 2.0. cDNA was synthesizedfrom 1 μg of RNA using miRNA First-Strand cDNA synthesis kit (AgilentTechnologies Inc.). qRT-PCR were analyzed for miR-455-3p, miR-4674,miR-3613-3p, miR-4668-5p, and mir-6722 as described previously.

Animal models. Amyloid-β transgenic (APP) mice were generated with themutant human APP gene 695-amino-acid isoform and a double mutation(Lys670Asn and Met671Leu) (35). The APP mouse model exhibitsage-dependent Aβ plaques as well as a distribution of Aβ plaques in thecerebral cortex and the hippocampus, but not in the striatum, the deepgray nuclei, and the brain stem. Disease in this mouse model parallelsAD in that elevated amounts of soluble Aβ correlate with increasedfree-radical production, and the Aβ plaques evoke a microglial reactionin their immediate vicinity. Cerebral cortex tissues were collected from6-month-old APP transgenic mice (n=6) and age-matched, non-transgenicwild-type mice (n=6). To determine transgene-positive mice to model thehuman APP, genotyping was performed in accordance with the TTUHSC Policyon Genotype Tissue Collection, using the DNA prepared from tail biopsyand PCR amplification (35). All mice were observed daily by a veterinarycaretaker and also examined twice weekly by laboratory staff. If anymice showed premature signs of neurological deterioration, they wereeuthanized before experimentation according to euthanasia procedureapproved by the TTUHSC-IACUC and were not used in the study.

Amyloid-β(1-42) treatment to cell lines. Human neuroblastoma (SH-SY5Y)and mouse neuroblastoma (N2a) cell lines were purchased from AmericanTissue Type Collection (ATCC) (Virginia, USA). Cells were grown in amedium (1:1) Dulbecco's modified eagle's medium and minimum essentialmedium, 5% fetal bovine serum, 1× penicillin and streptomycin) at 37° C.in a humidified incubator with a 5% CO₂ environment. After the cellswere seeded, they were allowed to grow for 24-48 hours or until 80%confluence in 6-well plates. They were then used for experimentation.Two different groups of cells were used: (1) untreated SH-SY5Y/N2a cellsand (2) Aβ(1-42) peptide treated SH-SY5Y/N2a cells. They were incubatedwith the Aβ(1-42) peptide (20 mM final concentration) in triplicate for6 hours. Both groups of cells were harvested after treatment andprocessed for total RNA extraction and miR-455-3p quantification.

MiRNAs pathway analysis. MiRNAs that were associated with signalingpathways were analyzed with the miRPath v3.0 web server algorithm (52).Briefly, species was defined as ‘human, mouse’ and miR-455-3p, miR-4674,miR-3613-3p, miR-4668-5p, and mir-6722 were entered. MiRNAs that targetgenes and biological pathways were analyzed, using microT-CDS andTarBase to classify the GO category, the P<0.05 of the KEGG pathwayenrichment, and the microT-CDS threshold (0.8). The miRNA-targeted genesin different KEGG molecular pathways were ranked according to theirP-value. The false discovery rate (FDR) P<0.05 was consideredstatistically significant.

Statistical analysis. The qRT-PCR validation analysis was based on the2{circumflex over ( )}-ΔΔCT value of genes in each sample from AD, MCIsubjects and controls. Statistical analysis was performed with Prismsoftware, v, 6 (La Zolla, CA). P-value was calculated, based on thepaired and unpaired t-tests for analyzing 2 groups and using one-waycomparative analysis of variance (ANOVA) when comparing between morethan 2 groups. P<0.05 was considered statistically significant.

Example 2

The goal of present study was to identify a suitable, non-invasive,blood-based early biomarker for AD detection. To achieve this goal, theinventors focused on circulatory microRNAs (cmiRNAs), which are quitestable in peripheral circulation and levels of particular miRNA seems tobe changing with disease severity. The previous research findings onhuman serum samples from AD patients, MCI individual and healthysubjects identified significant number of deregulated miRNAs in patientscompared to controls (Kumar et al., 2017). A few of them weresignificantly upregulated and some were down regulated in AD and MCIindividual compared to healthy controls. One of the most suitableidentified candidate in the study was microRNA-455-3p. Expression ofmiR-455-3p was found to be significantly upregulated in AD serumsamples, AD postmortem brains, AD mouse model, and AD cell lines (Kumaret al., 2017). Upregulation of miR-455-3p in different cell and mousemodels of AD proven its biomarker potential for AD. To furtherstrengthen the findings, the present study is focused on the ADpostmortem brains obtained from NIH NeuroBioBanks, human fibroblast, andB-lymphocytes cell lines derived from familial AD and sporadic ADpatients. Expression of miR-455-3p was quantified and its diagnosticpotential was examined in different sources. Further, in-silico analysiswas performed to understand the roles and downstream application ofmiR-455-3p in AD. Findings from this study, will provide the valuableinformation about miR-455-3p role in AD and in search of pre-clinicalbiomarker for early AD detection.

Study Subjects. (a) AD postmortem brains—Postmortem brains from ADpatients and healthy controls were obtained from three NIHNeuroBioBanks—(1) Human Brain and Spinal Fluid Resource Center, 11301Wilshire Blvd (127A), Los Angeles, Calif. (2) Brain Endowment Bank,University of Miami, Millar School of Medicine, 1951, NW 7th AvenueSuite 240, Miami, Fla. (3) Mount Sinai NIH Brain and Tissue Repository,130 West Kingsbridge Road Bronx, N.Y. Brain tissues were dissected fromthe Brodmann's Area 10 of the frontal cortices from AD patients (n=27)and age and sex matched healthy controls (n=15). Demographic andclinical details of study specimens were given in Table 5.

TABLE 5 Demographic and clinical details of the brain samples NeuroAutolysis S. No Sample ID Age Sex pathology Structure time 1 4130 67 FControl Broadmann's Area 10 11.8 2 4431 68 F Control Broadmann's Area 1023.7 3 4660 73 F Control Broadmann's Area 10 18.5 4 5072 83 M ControlBroadmann's Area 10 19.5 5 5190 68 M Control Broadmann's Area 10 20.3 6HCT15HAO1713 70 M Control Broadmann's Area 10 12.7 7 HCTZZC1711 82 FControl Broadmann's Area 10 14.2 8 HCT15HBC1709 83 M Control Broadmann'sArea 10 25 9 HCTZZT1702 84 M Control Broadmann's Area 10 15.5 10HCT15HBU1704 91 F Control Broadmann's Area 10 18.7 11 77428 65 M ControlBroadmann's Area 10 3.8 12 77431 103 F Control Broadmann's Area 10 3.813 77433 75 M Control Broadmann's Area 10 5 14 77436 93 M ControlBroadmann's Area 10 4.1 15 77437 84 F Control Broadmann's Area 10 5.4 164513 74 M AD Broadmann's Area 10 15.6 17 4498 76 M AD Broadmann's Area10 12.9 18 4204 68 M AD Broadmann's Area 10 11.9 19 4203 72 F ADBroadmann's Area 10 20.3 20 4454 82 F AD Broadmann's Area 10 9 21 404380 F AD Broadmann's Area 10 13 22 4382 74 F AD Broadmann's Area 10 16.223 4617 73 F AD Broadmann's Area 10 18.9 24 4718 93 F AD Broadmann'sArea 10 8.2 25 4608 80 M AD Broadmann's Area 10 3.1 26 4752 89 M ADBroadmann's Area 10 9 27 4788 65 M AD Broadmann's Area 10 7.8 28HBFR1703 69 F AD Broadmann's Area 10 22 29 HBFQ1711 77 M AD Broadmann'sArea 10 18 30 HBJG1710 79 M AD Broadmann's Area 10 23.8 31 HBDA1704 80 MAD Broadmann's Area 10 22.1 32 HCTYN1713 80 F AD Broadmann's Area 10 6.533 HBDI1710 85 F AD Broadmann's Area 10 8 34 HBEM1701 86 M ADBroadmann's Area 10 15.5 35 HBIP1701 90 F AD Broadmann's Area 10 22.1 36HBCG1703 90 F AD Broadmann's Area 10 8.5 37 HCTZX1702 95 M ADBroadmann's Area 10 19.8 38 77423 79 F AD Broadmann's Area 10 6.5 3977424 69 M AD Broadmann's Area 10 5.4 40 77425 75 M AD Broadmann's Area10 8 41 77426 94 F AD Broadmann's Area 10 4.3 42 77427 82 M ADBroadmann's Area 10 20.6

(b) AD patients cell lines—Human skin fibroblast and Lymphoblast cellculture systems were used for these studies. Banked skin fibroblasts andlymphoblast cells with the diagnoses AD, non-AD dementia (e.g.,Huntington's disease and Parkinson's disease, and schizophrenia), andage-matched control were obtained from the Coriell Institute of MedicalResearch, Camden, N.J., USA. The demographic details of cell lines alongwith their passage numbers, biopsy sources and tissue types wereprovided in Table 6. Cells were cultured and maintained in RPMI1640 forB-lymphocytes and MEM media for Fibroblasts (Life TechnologiesCorporation, NY, USA; supplemented with 10% Fetal Bovine Serum and 1×penicillin/streptomycin) at 37° C. with 5% CO₂ to the 90-100% confluencestage in 25 and 75 cm² cell culture flasks.

TABLE 6 Details of human Fibroblasts and B-lymphocytes Catalog PassageAge Biopsy Tissue Disease S. No. no no Sex (Years) sources type Racestatus (A) Fibroblasts 1 AG02261 11 M 61 Abdomen Skin Caucasian Healthycontrol 2 AG16104 6 F 55 Arm Skin Black Healthy control 3 AG16086 6 F 67Arm Skin Other Healthy control 4 AG12207 13 M 68 Arm Skin NA Healthycontrol 5 AG02258 6 F 46 Lung Lung Caucasian Healthy control 6 AG02262 4M 61 Lung Lung Caucasian Healthy control 7 AG06561 5 F 16FW# Sacrum SkinCaucasian Healthy control 8 AG12211 11 M 54 Lung Lung Caucasian Healthycontrol 9 AG05810 11 F 79 Arm Skin Caucasian Familial AD 10 AG06844 12 M59 Arm Skin Caucasian Familial AD 11 AG07613 16 M 66 Arm Skin CaucasianFamilial AD 12 AG09908 14 F 81 Arm Skin Caucasian Familial AD 13 AG0440019 F 61 Skin Skin Caucasian Sporadic AD 14 AG06263 11 F 67 Arm SkinCaucasian Sporadic AD 15 AG06264 7 F 62 Arm Skin NA Sporadic AD 16AG07375 6 M 71 Arm Skin Caucasian Sporadic AD 17 AG08243 7 M 72 Arm SkinCaucasian Sporadic AD 18 AG11368 15 M 77 Skin Skin Caucasian Sporadic AD(B) B-Lymphocytes 1 AG16639 na M 77 Peripheral Blood Caucasian Healthyvein control 2 AG11684 na M 82 Peripheral Blood Caucasian Healthy veincontrol 3 AG12034 na F 80 Peripheral Blood Caucasian Healthy veincontrol 4 AG11716 na M 98 Peripheral Blood Caucasian Healthy veincontrol 5 AG12032 na M 84 Peripheral Blood Caucasian Healthy veincontrol 6 AG16804 na F 90 Peripheral Blood Caucasian Healthy veincontrol 7 AG16927 na M 85 Peripheral Blood Caucasian Healthy veincontrol 8 AG16973 na F 80 Peripheral Blood Caucasian Healthy veincontrol 9 AG10673 na F 85 Peripheral Blood Black Healthy vein control 10AG16907 na F 88 Peripheral Blood Caucasian Healthy vein control 11AG08242 na M 72 Peripheral Blood Caucasian Familial vein AD 12 AG09905na M 72 Peripheral Blood Caucasian Familial vein AD 13 AG09907 na F 71Peripheral Blood Caucasian Familial vein AD 14 AG11755 na F 85Peripheral Blood Caucasian Familial vein AD 15 AG11757 na F 81Peripheral Blood Caucasian Familial vein AD 16 AG11758 na M 83Peripheral Blood Caucasian Familial vein AD 17 AG06204 na M 67Peripheral Blood Caucasian Sporadic vein AD 18 AG06868 na F 60Peripheral Blood Caucasian Sporadic vein AD 19 AG11366 na M 52Peripheral Blood Caucasian Sporadic vein AD 20 AG17512 na M 70Peripheral Blood African Sporadic vein American AD 21 AG17529 na F 86Peripheral Blood African Sporadic vein American AD 22 AG17574 na F 83Peripheral Blood African Sporadic vein American AD na* not available

Ethical Approval and Consent. The study was conducted at the GarrisonInstitute on Aging (GIA), Texas Tech University Health Sciences Center(TTUHSC), and study protocol was approved by the Institutional ReviewBoard of TTUHSC, Lubbock, Tex. for the use of biospecimens in ProjectFRONTIER (IRB #: L06-028). Regarding postmortem brains and cell linesused in the current study—each of the NIH NeuroBioBanks mentioned aboveoperated under their institution's IRB approval. As determined by theFDA Research Involving Human Subjects Committee, current study did notreach the definition of “Human Subject Research” at 45 CFR 46.102(f) andthus, 45 CFR Part 46 does not apply (Ferguson et al., 2017). Further,according to Office for Human Research Protections Guidelinesbiospecimens obtained by the researchers from NIGMS Human Genetic CellRepository are not considered to be human subjects because conductingresearch with the samples does not involve an intervention orinteraction with the individual and the samples do not containidentifiable private information (www.coriell.org).

RNA Extraction—Total RNA was isolated from the 80 mg of frontal corticesusing the TriZol RT reagent (Ambion, USA) as per manufacturerinstructions. Briefly, tissue samples were homogenized in 1 ml of TriZolreagent with Bio-Gen PRO200 Homogenizer (PRO Scientific Inc., CT, USA)in a 2-ml RNase-free tube. Chloroform (0.2 ml) was added to the tissuehomogenate, vigorously shaken for 15 s, and stored for 5 min at roomtemperature. The mixture was then centrifuged at 12,000 g for 15 min at4° C. The supernatant was transferred to a new tube and precipitatedwith 0.5 ml of isopropanol for 15 min at room temperature. Samples werecentrifuged at 12,000 g for 10 min at 4° C. The resulting RNA pellet waswashed with 1 ml of 75% ethanol and centrifuged at 7,500 g for 5 min at4° C. The RNA pellet was dried and dissolved in 50 μl of DEPC-treatedwater. The quality and quantity of the RNA were analyzed by NanoDropanalysis. The value of absorbance of each brain RNA sample (A260/A280)was 1.8-2.0.

Quantification of miRNAs Expression by Quantitative Real-TimePCR-Quantification Involved Three Steps:

Polyadenylation—One microgram of total RNA was polyadenylated with anmiRNA First-Strand cDNA synthesis kit (Agilent Technologies Inc., CA,USA), following manufacturer's instructions. Briefly, a polyA reactionwas prepared by mixing RNA with 4.0 μl of 5× poly A polymerase buffer,1.0 μl of rATP (10 mM), 1 μl of E. coli poly A polymerase, producing afinal volume of 20 μl with RNase free water. The tube with thesecomponents was incubated at 37° C. for 30 min, followed anotherincubation at 95° C. for 5 min to terminate the adenylation reaction(Kumar et al., 2014).

cDNA synthesis—Ten microliters of polyadenylated miRNAs were processedfor cDNA synthesis with the miRNA First-Strand cDNA synthesis kit(Agilent Technologies Inc.). The following reaction components werecombined in a tube: 2 μl of 10× AffinityScript RT buffer, 0.8 μl of dNTPmix (100 mM), 1 μl of RT adaptor primer (10M), 1.0 μl of AffinityScriptRT/RNase Block enzyme, and polyadenylated RNA. The combination resultedin a reaction volume of 20 μl RNase-free water. This reaction mixturewas incubated at 55° C. for 5 min, then at 25° C. for 15 min, followedby an incubation at 42° C. for 30 min, and a final incubation at 95° C.for 5 min in a Veriti 96 well thermal cycler (Applied Biosystems, USA).Resulting cDNAs were diluted with 20 μl of RNase-free water and storedat 80° C. for further analysis.

Real-time RT-PCR—Real-time RT-PCR reaction was performed by preparing areaction mixture containing 1 μl of miRNA-specific forward primer (10m), 1 μl of a universal reverse primer (3.125 m) (Agilent TechnologiesInc., CA, USA), 10 μl of 2×SYBR Green PCR master mix (AppliedBiosystems, NY, USA), and 1 μl of cDNA. To this mixture RNase-free waterwas added up to a 20 μl final volume. Primers for hsamiR-455-3p(Forward: 5′ GCAGTCCATGGGCATATACAC-3′ (SEQ ID NO: 1), and U6snRNA (P1:5′-GCTTCGGCAGCACATATACTAA-3′ (SEQ ID NO: 8) and Reverse:5′-TATGGAACGCTTCACGAATTTGC-3′ (SEQ ID NO: 9)) were synthesizedcommercially (Integrated DNA Technologies, Inc. Iowa USA). To normalizethe miRNA expression, U6 snRNA (small nuclear RNA) expression was alsoquantified in the tissue and cells, which was used as an internalcontrol. The reaction mixture of each sample was prepared intriplicates. The reaction was set in the 7900HT Fast Real Time PCRSystem (Applied Biosystems, USA) using following reaction conditions:initial denaturation at 95° C. for 5 min, denaturation at 95° C. for 10s, annealing at 60° C. for 15 s, and extension at 72° C. for 25 s. Therelative levels of miR-455-3p in the AD patients vs. the controlssubjects were determined in terms of their fold change, using theformula (2-11Ct), where 1Ct was calculated by subtracting Ct of U6snRNAfrom the Ct of miR-455-3p. Real-time RT-PCR was performed in triplicate,and the data were expressed as the mean±SD (Kumar et al., 2014; Hamam etal., 2016).

In-Silico Analysis for miR-455-3p. MiR-455-3p target genes were analyzedusing various on-line miRNA algorithms (diana-microt, microrna.org,mirdb, rna22-has, targetminer, and targetscan-vert). Details aboutpredictive and validated transcripts were obtained by searchinghsamiR-455-3p.1 and hsa-miR-455-3p.2 isoforms. Target genes were checkedfor following parameters: (i) their representative transcripts, (ii)number of 3P-seq tags supporting UTR +5, (iii) link to sites in UTRs,(iv) conserved sites/poorly conserved sites, (v) cumulative weightedcontext++ score, (vi) total context++ score, and (vii) aggregate PCT(preferentially conserved targeting) values. Further, predictedconsequential pairing showed the miRNA-target complementarity at insideor outside the seed regions of miRNAs was checked at untranslatedregions links (http://www.targetscan.org).

Statistical Analysis. The real-time RT-PCR data was analyzed by usingthe formula 2-ACT value of genes in each sample from AD patient'ssamples and controls. Statistical analysis was performed with Prismsoftware, v, 6 (La Zolla, CA). P-value was calculated, based on theunpaired t-tests for analyzing two groups and using one way comparativeanalysis of variance (ANOVA) when comparing between more than twogroups. ROC curve was plotted based on the 1CT value of samples inpatients and control groups. Correlation analysis was performed usingtwo tailed Pearson correlation coefficient (r) calculation considering95% confidence interval. P<0.05 was considered statisticallysignificant.

Up Regulation of miR-455-3p Expression in AD. AD Postmortem Brains—TotalRNA was extracted from the postmortem brains of healthy controls (n=15)and AD patients (n=27) and expression of hsa-miR-455-3p was quantifiedby real-time RT-PCR analysis. Fold-change was calculated based on the1CT value of miR-455-3p in AD patients' vs. healthy controls. The (ΔCT)value (mean±SD) was significantly (P=0.0001) higher in AD patients(−6.89±0.21) compared to the healthy controls (−8.94±0.56; FIG. 10A).Interestingly, fold-change analysis indicated the significantly higherexpression of miR-455-3p in AD patients.

AD Fibroblasts—Similarly, expression of miR-455-3p was quantified in theskin fibroblast cells generated form familial AD patients (n=4),sporadic AD patients (n=6), and healthy control subjects (n=8).Differences in ACT values was evaluated among three groups using one-wayANOVA. Results showed the higher (−ΔCT) values (mean±SD) of miR-455-3pin familial and sporadic patients compared to controls. However,significant difference (P=0.014) in (−ΔCT) value was observed insporadic cases (˜7.35±1.39) compared to control samples (˜9.37±0.76;FIG. 10B). AD B-Lymphocytes—Further, the inventors checked the level ofmiR-455-3p in B-lymphocytes obtained from familial AD patients (n=6),sporadic AD patients (n=6), and healthy controls (n=10). The (−ΔCT)(mean±SD) value was compared among three group using one-way ANOVA.Analysis showed the variations in miR-455-3p level among these groups,however significant difference (P=0.044) in (−ΔCT) value was reportedbetween sporadic AD cases (−13.98±0.73) and controls (−15.50±0.80; FIG.10C). Hence, results obtained from AD postmortem brains, AD fibroblast,and AD B-lymphocyte were conclusively confirmed the decisive role ofmiR-455-3p in AD assessment.

Receiver Operating Characteristics Curve Analysis of miR-455-3p. ADPostmortem Brains—To determine the diagnostic performance of miR-455-3pexpression in AD patients, ROC curve was plotted using (ΔCT) values ofmiR-455-3p in AD patients and healthy controls. Analysis showed thesignificant area under ROC curve (AUROC) value of miR-455-3p(AUROC=0.792) with the 95% confidence interval was 0.637-0.948(P=0.0018). The cut-off value was 8.16 with sensitivity of 88.89% (95%confidence interval: 70.84-97.65%) and specificity was 66.67% (95%confidence interval: 38.38-88.18%) in AD brain samples compared withhealthy controls (FIG. 11A).

In AD Fibroblasts—ROC curve was analyzed for miR-455-3p expression infibroblast cells form familial and sporadic AD patients vs. healthycontrols. However, significant AUC value was obtained for ROC curve whencomparing between sporadic AD patients with healthy controls. AUROCvalue was (0.861) with 95% confidence interval of 0.6036-1.119(P=0.037). The cut-off value was 9.12 with sensitivity of 83.33% (95%confidence interval: 35.88-99.58%) and specificity was also 66.67% withconfidence interval (22.28-95.67%; FIG. 11B).

In AD B-Lymphocytes—Similarly ROC curve for miR-455-3p was analyzed inB-lymphocytes of AD patients. Analysis between sporadic AD patients andhealthy controls showed the fair AUROC value (0.722) with 95% confidenceinterval of 0.4185-1.026 (P=0.20). The cut-off value was 14.90 withsensitivity of 66.67% (95% confidence interval: 22.28-95.67%) andspecificity was 50.00% (95% confidence interval: 11.81-88.19%; FIG.11C). Thus, analysis showed that ROC analysis of miR-455-3p inB-lymphocytes was not significant. However, data from postmortem ADbrains and AD fibroblasts cells showed significant ROC curve datafurther confirmed that the miR-455-3p as a valuable molecule capable ofdiscriminating the patients with AD from healthy individuals.

Correlation of miR-455-3p Expression with Patients' Demographic Data.The inventors analyzed miR-455-3p expression levels in relation to (1)postmortem interval, (2) AD patients' age, and also (3) donors' age offibroblasts, and (4) B-lymphocytes using Pearson correlationcoefficients (r). AD postmortem brains showed a negative correlationr=−0.146 (with 95% confidence interval: −0.498 to 0.247; P=0.466)between brains postmortem interval and miR-455-3p expression level (FIG.12A). Whereas a positive correlation r=0.355 (with 95% confidenceinterval: −0.029 to 0.647; P=0.069) was observed between the age of ADpostmortem brains and miR-455-3p level (FIG. 12B). However, P-valueswere not significant in both cases. Thus, results showed a trend ofreduced levels of miR-455-3p with increased postmortem interval andincreased trend of miR-455-3p with patients' age. As shown in FIG. 12C,FIG. 12D, donors' age for fibroblasts (r=−0.396, 95% confidenceinterval: −0.821 to 0.310; P=0.256), and B-lymphocytes (r=0.235, 95%confidence interval: −0.391 to 0.713; P=0.461), the inventors did notfind statistical significance, between donors age with miR-455-3p levelsfor fibroblasts and B-lymphocytes, indicating that donors age do notaffect miR-455-3p expression levels.

In-Silico Analysis for miR-455-3p Function in AD. In-silico analysis wasperformed to understand the functions of miR-455-3p and its possiblerole in AD pathogenesis. Analysis was performed using the variousbio-informatics algorithms such as DIANA-MICROT, MICRORNA.ORG, MIRDB,RNA22-HAS, TARGETMINER, and TARGETSCAN-VERT. As per miRbase database, atotal of 3,102 reads of miR-455-3p has been detected by deep sequencingin 62 experiments (www.mirbase.org). Each algorithm was run formiR-455-3p and validated/predictive target genes were analyzed. A totalof 323 predicted transcripts/human genes were identified with conservedmiR-455-3p.2 binding site. Out of these genes most potential 13 targetsgenes were screened for those were having the roles in AD pathogenesis.Important ones were: APP, NGF, USP25, PDRG1, SMAD4, UBQLN1, SMAD2, TP73,VAMP2, HSPBAP1, and NRXN1 (Table 7). miR-455-3p having at least one ortwo binding site at 3′UTR of the genes and total context++score rangesfrom −0.1 to −0.46. For e.g., miR-455-3p binds at the two sequence sitesof 3′ UTR of APP gene at sequence position 522-528 and 3,139-3,145.Interaction of miR-455-3p at these sites will influence the expressionlevel of APP genes. Hence, these analyses indicated the possible way themiR-455-3p involved in AD pathogenesis.

TABLE 7 Predictive/validated gene targets of miR-455-3p involved in ADOrtholog of 3P- Conserved Cumulative Aggre- Representative targetRepresentative seq sites weighted Total gate S. No miRNA gene transcriptGene name tags + 5 total context++score context++score PCT 1 hsa-miR-NGF ENST00000369512.2 nerve 27 1 −0.46 −0.46 0.38 455-3p.2 growth factor(beta polypeptide) 2 hsa-miR- USP25 ENST00000285681.2 ubiquitin 2012 2−0.45 −0.45 0.6 455-3p.2 specific peptidase 25 3 hsa-miR- PDRG1ENST00000202017.4 p53 and 116 1 −0.45 −0.45 <0.1 455-3p.2 DNA-damageregulated 1 4 hsa-miR- SMAD4 ENST00000398417.2 SMAD 403 2 −0.3 −0.32<0.1 455-3p.2 family member 4 5 hsa-miR- UBQLN1 ENST00000376395.4ubiquilin 1 471 2 −0.3 −0.33 <0.1 455-3p.2 6 hsa-miR- APPENST00000346798.3 amyloid beta 4570 2 −0.29 −0.35 <0.1 455-3p.2 (A4)precursor protein 7 hsa-miR- SMAD2 ENST00000262160.6 SMAD 1196 2 −0.2−0.28 0.33 455-3p.1 family member 2 8 hsa-miR- TP73 ENST00000378280.1tumor 831 1 −0.14 −0.14 0.3 455-3p.1 protein p73 9 hsa-miR- VAMP2ENST00000316509.6 vesicle- 1840 1 −0.11 −0.11 0.26 455-3p.1 associatedmembrane protein 2 (synaptobrevin 2) 10 hsa-miR- HSPBAP1ENST00000383659.1 HSPB (heat 22 1 −0.11 −0.15 <0.1 455-3p.1 shock 27kDa) associated protein 1 11 hsa-miR- NRXN1 ENST00000342183.5 neurexin 15 1 −0.1 −0.1 0.3 455-3p.1

The purpose of the study was to determine the blood based peripheralbiomarkers for AD. The inventors recently conducted a high throughputmicroRNA analysis using serum-derived RNA samples from MCI subjects, ADpatients, and healthy control subjects (Kumar et al., 2017). Theinventors found several differentially expressed miRNAs in MCI subjectsand patients with AD relative to healthy controls. Further, theinventors verified differentially expressed miRNAs using real-timeRT-PCR from serum-derived miRNAs, and also from cell and mouse models ofAD. In the current study, the inventors extended the investigationsusing large numbers of fibroblasts, B-lymphocytes from familial andsporadic AD patients and age-matched control subjects. The inventorsfound miR-455-3p levels were upregulated in the fibroblasts andB-lymphocytes from AD patients relative to healthy control subjects.However, a significant difference was observed in the cells formsporadic AD patients compared to healthy controls. Similarly, inB-lymphocytes, miR-455-3p level was significantly upregulated insporadic AD cases compared to controls (P=0.044). Receiver operatingcharacteristic curve analysis indicated the significant area under curvevalue of miR-455-3p in AD postmortem brains (AUROC=0.792; P=0.001) andAD fibroblasts cells (AUROC=0.861; P=0.03). These observations show thatmiR-455-3p is a biomarker for sporadic AD.

An early stage pre-clinical diagnostic biomarkers are urgently needed todetect disease process early on in life and take necessary action toprevent and/or delay disease progression. Recent molecular biologystudies using serum/plasma revealed that several circulatory microRNAscan be used as potential peripheral biomarkers for AD (Kumar and Reddy,2016). However, these circulatory microRNAs are needed furthervalidation using postmortem AD brains and cell and mouse models of AD.Therefore, more accurate and mechanistic research is needed to determinepotential candidates as biomarkers for AD. As mentioned above, therecent lab study on AD serum samples and other AD sources/AD mouse modelunveiled the miR-455-3p as potential biomarker candidate for AD. Manyother reports identified the role of miR-455-3p in several cancers andchondrogenic differentiation (Chen et al., 2016; Cheng et al., 2016; Liet al., 2016; Liu et al., 2016; Qin et al., 2016; Zheng et al., 2016;Zhao et al., 2017). The study was the first to reveal the higherexpression level of miR-455-3p in persons with AD.

Current study is the continuation of the ongoing biomarkers researchproject in the Reddy Lab. Here, first the inventors investigatedmiR455-3p levels in the well-defined postmortem brain tissues from ADpatients. All tissues were dissected from the affected area (Broadmann'sarea 10) of AD patients and commonly used for the investigation of ADpathogenesis (Wilcock et al., 2015; De Rossi et al., 2016; Shackleton etal., 2017). The current study on AD postmortem findings revealed thatmiR-455-3p levels are significantly increased in a large number (n=27)of AD brains and a significant AUC value also strengthen its biomarkerpotential. However, the inventors don't know the exact reason for theupregulation of miR-455-3p in AD brains and further, the inventors stilldo not know molecular mechanism(s) of its increased levels. By way ofexplanation, and in no way a limitation of the present invention, andeased on current findings, the upregulation of miR-455-3p—may be acompensatory to the amyloid beta toxicity in disease process.

Beside brain tissues, the inventors also investigated the AD fibroblastsand B-lymphocytes for miR-455-3p expression. These AD cell lines are thegood sources for the investigation of AD pathologies and associatedmolecular changes in the patients' genome (Khan and Alkon, 2016). Bothcell types showed the significantly higher levels of miR-455-3p,especially in sporadic AD cases but not in familial AD. Further, highlevel of miR-455-3p in AD fibroblasts and lymphoblasts indicate thatincreased levels of miR-455-3p is a typical feature of AD—both in thebrain and peripheral cells. Alteration of miR-455-3p expression in ADcell lines indicates the strong molecular association of miR-455-3p withAD progression.

In order to expose the roles and functions of miR-455-3p in AD,in-silico analysis provides the valuable information. As described 11genes were reported to involve in AD progression (FIGS. 3A-E) (Burton etal., 2002; Li et al., 2004; Slifer et al., 2006; T6th et al., 2013;Sindi et al., 2014; Jung et al., 2015, 2016; Malkki, 2015; Vallortigaraet al., 2016; Kuruva et al., 2017). To understand the roles ofmiR-455-3p in AD, expression of these genes needs to be studied by usingmiR-455-3p modulation approaches (mimics/inhibitors). In this direction,next phase of the study is to determine the effect of miR-455-3p on itsAD related target genes. Current focus of the laboratory is tounderstand the role of miR-455-3p in APP processing and amyloid betamodulation, using miR-455-3p mimics and inhibitor treatments. Theinventors also predict that two potential binding sites of miR-455-3p atthe 3′UTR of APP gene may be involved in the modulation of full lengthAPP. Further, the inventors also predict that miR-455-3p affects the APPprocessing and amyloid beta production.

In summary, for the first time, the inventors report that microRNA455-3pis a peripheral biomarker for AD. These findings are based on (1)blood-based circulatory microRNAs from AD patients, (2) AD postmortembrains, AD cell lines, and AD mouse models and a large number of ADfibroblasts and lymphoblasts.

Example 3

The purpose of this example was to understand the protective role ofmiR-455-3p against abnormal amyloid precursor protein (APP) processing,amyloid beta (Aβ) formation, defective mitochondrial biogenesis/dynamicsand synaptic damage in AD progression. In-silico analysis of miR-455-3phas identified the APP gene as a putative target. Using mutant APPcells, miR-455-3p construct, biochemical and molecular assays,immunofluorescence and transmission electron microscopy (TEM) analyses,the inventors studied the protective effects of miR-455-3p on −1) APPregulation, amyloid beta (Aβ)(1-40) & (1-42) levels, mitochondrialbiogenesis & dynamics; 3) synaptic activities and 4) cell viability &apoptosis. A luciferase reporter assay confirmed the binding ofmiR-455-3p at the 3′UTR of APP gene. Immunoblot, sandwich ELISA andimmunostaining analyses revealed that the reduced levels of the mutantAPP, Aβ(1-40) and Aβ(1-42), and C99 by miR-455-3p. It was also found thereduced levels of mRNA and proteins of mitochondrial biogenesis (PGC1a,NRF1, NRF2, and TFAM) and synaptic genes (synaptophysin and PSD95) inmutant APP cells; on the other hand, mutant APP cells that expressmiR-455-3p showed increased mRNA and protein levels of biogenesis andsynaptic genes. Additionally, expression of mitochondrial fissionproteins (DRP1 and FIS1) were decreased while the fusion proteins (OPA1,Mfn1 and Mfn2) were increased by miR-455-3p. TEM analysis showed adecrease in mitochondria number and an increase in the size ofmitochondrial length in mutant APP cells transfected with miR-455-3p.Based on these observations, it is demonstrated here that miR-455-3pregulates APP processing and is protective against mutant APP-inducedmitochondrial and synaptic abnormalities in AD.

The inventors identified several potential genes that are targeted bymiR-455-3p and very important in AD pathogenesis. APP gene was one ofthe top-targeted genes in the miRNA analysis. The precise molecularlinks between miR-455-3p and APP gene are now described, particularlywhether upregulated miR-455-3p is protective or deleterious in theprogression and pathogenesis of AD and 2) further, the impact of 455-3pon APP processing & Aβ production, mitochondrial biogenesis,mitochondrial dynamics and synaptic activity in AD neurons is unclear.To address these questions, APP processing, mitochondrial biogenesis,mitochondrial dynamics, mitochondrial morphology, synaptic activity andcell viability in mutant APP cells that express miR-455-3p was studied.

Cell culture. The N2a mouse neuroblastoma cell line was purchased fromthe ATCC (American Type Culture Collection) and was maintained in theReddy laboratory. The cells were grown in DMEM+F12 (1:1) supplementedwith 10% FBS at 37° C. with 5% CO₂.

Mutant APP cDNA construct. The mutant APP Swe cDNA clone (pCAX-APPSwe/Ind) was obtain from the laboratory of Dr. Arubala Reddy [32]. APPSwe cDNA was cloned into a mammalian expression vector pRP-Puro-CAG. ThepRP vector is a puc backbone having a CMV promoter and an SV40polyadenylation site with puromycin selection for stable transfection.The sequence output was confirm with the NCBI sequence hAPP[NM_201414.2]*(K595N M596L V642F), relevant sequence incorporated hereinby reference.

MiR-455-3p expression vector. MiR-455-3p expression vector(pRP[Exp]-U6>hsa-miR-455-3p-CAG-EGFP) was purchased from VectorBuilder(Cyagen Biosciences, Santa Clara, Calif., USA). It was a 5128 bp plasmidpropagated in Stbl3 host and contained a Green Fluorescent Protein (GFP)encoding gene (data not shown). GFP expression readily allowed thedetection and confirmation of transfection.

Target prediction analysis of miR-455-3p. In silico analysis wasperformed with various bio-informatics algorithms, such as DIANA-MICROT,MICRORNA.ORG, MIRDB, RNA22-HAS, TARGETMINER, and TARGETSCAN-VERT.MiR-455-3p had at least 2 binding sites at 3′UTR of the genes and totalcontext++ score ranges from −0.1 to −0.46. For example, miR-455-3p bindsat the 2 sequence sites of 3′ UTR of the APP gene at sequence positions522-528 and 3,139-3,145 (FIG. 13A).

FIGS. 13A to 13H—MiR-455-3p interaction with the APP gene and itseffects on neuroblastoma cells survival. (FIG. 13A) The two putativebinding sites of miR-455-3p at the 3′-UTR regions of a wild-type APPgene in humans and mice. Region A (522-528) had 7 nucleotide bindingsites, and Region B (3139-3145) had 6 nucleotide binding sites with theseed sequence of miR-455-3p in both the human and mouse wild-type APPgene (shown in green). (FIG. 13B) Luciferase reporter assay formiR-455-3p binding with the APP gene. Normalized luciferase activity(Firefly/Renilla) in neuroblastoma cells co-transfected with the APP3′UTR clone (HmiT009578-MT06) and miR-455-3p mimics. Firefly/Renillaluciferase activity of the APP 3′UTR clone was significantly decrease bythe miR-455-3p. (FIG. 13C) Representative images of neuroblastoma cellsmorphology after 24 hr of miR-455-3p mimics and inhibitor transfection(10× magnification). Quantitative measurement of miR-455-3p (FIG. 13D),and APP fold-change (FIG. 13E), in cells at 24 hr post mimics andinhibitor transfection. (FIG. 13F) Representative images of Annexin Vand PI staining of cells at 24 hr post-transfection of miR-455-3p mimicsand inhibitor. White arrow represents the viable (green) and dead (red)cells. Percentage (%) of apoptotic cells (FIG. 13G), and viable cellspopulations (FIG. 13H), after 24 hr of miR-455-3p mimics and inhibitortransfection. (*P<0.05) (**P<0.01) (***P<0.001)

Luciferase reporter assay. To confirm the binding of miR-455-3p at the3′UTR of the APP gene, neuroblastoma cells were plated at a density of5×10⁴ cells per well in a 24-well plate for one day before transfection.The cells were then transfected with APP 3′UTR target expression clone(HmiT009578-MT06) for the human APP (NM_000484.2) and miRNA target clonecontrol vector having mutated 3′UTR of APP gene pEZX-MT06(CmiT000001-MT06) (Genecopoeia, Rockville, Md., USA). Cells were alsotransfected with miR-455-3p mimics and mimic controls (AppliedBiological Materials, Inc., Richmond, BC V6V 2J5, Canada). After 24 hrof transfection, luciferase activity was measured with the Luc-Pair™Duo-Luciferase Assay Kit 2.0 (Genecopoeia, Rockville, Md., USA) as permanufacturer instructions. The samples were read with an illuminometer.

Transfection of miR-455-3p mimics and inhibitors. Neuroblastoma cellswere transfected with 25 nM (final concentration) of miR-455-3p mimicoligonucleotide and miR-455-3p inhibitor oligonucleotide (AppliedBiological Materials, Richmond, BC, CANADA) using Lipofectamine™ 2000reagent (Invitrogen Life Technologies, Carlsbad, Calif., USA) followingthe manufacturer's instructions.

Transfection of the MiR-455-3p vector and the mutant APP cDNA.Neuroblastoma cells were cultured in antibiotic-free media, in 6-wellplates one day before transfection. On the next day, when cells reached70% to 80% confluence, they were transfected with the miR-455-3p vectorand the mutant APP cDNA with the Lipofectamine™ 2000 reagent. Mutant APPcDNA contains three identical sequences at three different locationsthose were complementary to the seed sequence of miR-455-3p.

Apoptotic assay. A cell-based apoptosis assay was performed, using theCellometer Vision CBA Image Cytometry System (Nexcelom Bioscience, LLC,Lawrence, Mass., USA) with 2 fluorophores—Annexin V-FITC and propidiumiodide (PI) staining solutions, following manufacturer's instructions.Briefly, neuroblastoma cells were harvested using trypsin, then spundown to 300 g for 3 min. The pellets were washed with 1×PBS. Cells werecounted using a hematocytometer, and 100,000 to 150,000 cells werecollected and resuspended in 40 μl of an Annexin V binding buffer. Fiveμl of Annexin V-FITC reagent (green) and PI (red) each were added to thebinding buffer containing the cells. The solution was gently mixed bypipetting up and down. It was then incubated for 15 min at roomtemperature in the dark. After incubation, 250 μl of 1×PBS was added andspun down at 300 g for 3 min. Cell pellets were re-suspend in 50 μl ofan Annexin V binding buffer and assessed for apoptosis analysis [32].

Cell viability test (MTT assay). Mitochondrial respiration, an indicatorof cell viability, was assessed in the neuroblastoma cells from controland experimental treatments (n=4), using the mitochondrial-dependentreduction of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) to formazan as described in Reddy et al, (2016) [33].Cells were trypsinized until they have lifted off the cell cultureplate, then spin down at 300 g for three minutes, decant the supernatantand re-suspend cells in 1×PBS. 20 μl of cells were transferred to thecountering chamber, cell viability was determined by using theCellometer Vision CBA Image Cytometry System.

Assessment of miR-455-3p levels—Quantification of miR-455-3p involved 3steps:

(i) Polyadenylation. One g of total RNA was polyadenylated with a miRNAFirst-Strand cDNA synthesis kit (Agilent Technologies Inc., CA, USA),following manufacturer's instructions [18].

(ii) cDNA synthesis. Ten L of polyadenylated miRNAs were processed forcDNA synthesis with the miRNA First-Strand cDNA synthesis kit (AgilentTechnologies Inc., CA, USA). Resulting cDNAs were diluted with 20 μL ofRNase-free water and stored at −80° C. for further analysis.

(iii) Real-time qRT-PCR. Real-time RT-PCR was performed by preparing areaction mixture containing 1 τL of miRNA-specific forward primer (10μm), 1 τL of a universal reverse primer (3.125 μM) (Agilent TechnologiesInc., CA, USA), 10 μl of 2×SYBR Green PCR master mix (Applied BiosystemsCA), and 1 μL of cDNA. To this mixture RNase-free water was added up toa 20-μL final volume. Primers used in the current study were synthesizedcommercially (Integrated DNA Technologies, Inc., City, Iowa, USA). Tonormalize the miRNA expression, snoRNA-202 (small nuclear RNA)expression was also quantified in the cells, which was used as aninternal control. The reaction mixture for each sample prepared intriplicates was set in the 7900HT Fast Real-Time PCR System (AppliedBiosystems). MiRNA fold change were calculated by using the formula(2^(−ΔΔct)) [18, 21].

Messenger RNA levels of APP, mitochondrial biogenesis and synapticgenes. Quantification of mRNA levels in APP, mitochondrial biogenesis,and synaptic genes was carried out with real-time RT-PCR using methodsdescribed in Reddy et al, (2016) [33]. The oligonucleotide primers weredesigned with primer express software (Applied Biosystems) for thehousekeeping genes β-actin, APP, PGC1a, NRF1, NRF2, TFAM, DRP1, FIS1,OPA1, Mfn1, Mfn2, synaptophysin, and PSD95. SYBR-Green chemistry-basedquantitative real-time RT-PCR was used to measure mRNA expression ofthese genes using β-actin and GAPDH as housekeeping genes, as describedby Manczak et al, (2016) [34].

Immunoblot analysis for the mutant APP protein. Immunoblot analysis wasperformed, using protein lysates prepared from all groups of cells(cells transfected and untransfected with mutant APP, miR455-3p), usingthe 6E10 antibody that recognizes full-length mutant human APP and Aβ.The inventors also performed immunoblot analysis for all mitochondrialbiogenesis proteins. Twenty μg of protein lysates were resolved on a4-12% Nu-PAGE gel (Invitrogen). The resolved proteins were transferredto nylon membranes (Novax Inc., San Diego, Calif., USA) and thenincubated for 1 hr at room temperature with a blocking buffer (5% drymilk dissolved in a TBST buffer). The nylon membranes were incubatedovernight with the primary antibodies. The membranes were washed with aTBST buffer 3 times at 10-min intervals and then incubated for 2 hr withan appropriate secondary antibody, sheep anti-mouse HRP 1:10,000,followed by three additional washes at 10-min intervals. Proteins weredetected with chemiluminescence reagents (Pierce Biotechnology,Rockford, Ill., USA), and the bands from the immunoblots were visualized[32].

Immunostaining and immunofluorescence analysis. To determine theimmunoreactivity and intensity of the APP protein, the inventorsperformed immunofluorescence analysis using neuroblastoma cellstransfected with a miR-control vector, a miR-455-3p vector, and a mutantAPP cDNA. The cells were co-transfected with an APP clone and amiR-455-3p vector. The cells were grown on coverslips for 24 hrpost-transfection. They were then were washed with 1×PBS three times for5 min each and fixed in freshly prepared 4% paraformaldehyde in PBS for10 min. The cells were washed again with PBS and permeabilized with 0.1%Triton-X100 in PBS. The cells were blocked with a 1% blocking solution(Invitrogen) for 1 hr at room temperature. The cells were incubated with6E10 primary antibody (1:200 dilution) (Biolegend, San Diego, Calif.)overnight at 4° C. After incubation, the cells were washed 3 times withPBS, for 10 min each. The cells were incubated with an anti-mousesecondary antibody conjugated with Fluor 488 (1:500 dilution)(Invitrogen) for 1 hr at room temperature, then washed 3 times with PBS,and mounted on slides. Photographs were taken with a multiphoton laserscanning microscope system (ZeissMeta LSM510). To quantify theimmunoreactivities of antibodies, 10-15 photographs were taken at 20×magnification. Statistical significance was assessed by the intensitiesof red, green or blue, using NIH ImageJ software [32].

ELISA for Aβ₍₁₋₄₀₎ and Aβ₍₁₋₄₂₎. The protein lysates were prepared fromneuroblastoma cells pellets and soluble Aβ levels were measured, usinghuman-specific Aβ(1-40) and Aβ(1-42) sandwich ELISA kit. The inventorsused Human Quantikine® ELISA Amyloid 3 (aa1-40) and (aa1-42) immunoassaykit as per manufacturer instructions (R&D Systems, Inc. Minneapolis,Minn., USA).

Transmission electron microscopy. Experiments were performed in 60-mmPetri dishes. Neuroblastoma cells were transfected with a miR-controlvector, the miR-455-3p vector, and the mutant APP cDNA; and wereco-transfected with the APP clone and the miR-455-3p vector. After 48hr, the cells were washed with 5 ml 1×PBS. Cell pellets were dissolvedin a fixative solution (8% glutaraldehyde, 16% paraformaldehyde, and0.2M sodium cacodylate buffer) for 1 hr at room temperature. The cellswere removed from the fixative solution and scraped into one ml of afresh fixative solution. Cells were incubated at room temperature for 30min. The cells were centrifuge at 300 g for 3 min [32]. The resultingcell pellet was undergone electron microscopy at the Imaging CoreFacility at Texas Tech University.

Statistical considerations. Statistical analyses were conducted, byusing the student T-test for analyzing 2 groups of samples, and one-waycomparative analysis of variance (ANOVA) was used for analyzing morethan two groups of samples. Significant differences in five group ofsamples were calculated by Bonferroni's multiple comparison tests.Statistical parameters were calculated, using Prism software, v6 (LaZolla, CA, USA). P<0.05 was considered statistically significant.

MiR-455-3p interaction with the APP gene. The inventors in silico miRNAtarget prediction analysis showed two binding sites of miR-455-3p at 3′UTR of the APP gene (ENST00000346798.3) at sequence positions 522-528and 3,139-3,145 (FIG. 13A). The cumulative weighted context++ score was−0.29, total context++ score was −0.35, and the aggregate PCT was <0.1for miR-455-3p (Table 8). The miR-455-3p binding sequence at the 3′UTRof APP mRNA was similar in the human and mouse genome. To confirm theseresults, a luciferase reporter assay was performed on a vector having awild-type APP 3′UTR sequence and on a control vector. Luciferaseactivity was significantly (P=0.002) reduced in the cells transfectedwith miR-455-3p mimics compared to their respective controls (FIG. 13B).These results indicate that the 3′UTR site of mRNA from the APP proteinconsists of conserved binding sites for miR-455-3p.

TABLE 8 Parameters for miR-455-3p binding at 3′UTR of APP gene PreviousTargetScan publication(s) None Aggregate PCT <0.1 Total context ++ score−0.35 Cumulative weighted context ++ score −0.29 Representative miRNAhsa-miR-455-3p.2 6mer sites 1 Poorly conserved 7mer-A1 sites 1 Poorlyconserved 7mer-m8 sites 1 Poorly conserved sites total 2 3P-seq tags + 54570 Gene name amyloid beta (A4) precursor protein Representativetranscript ENST00000346798.3 Target gene APP

MiR-455-3p functional analysis. (a) Neuroblastoma cells morphologytreated with miR-455-3p mimics and inhibitors. Since miR-455-3p targetAPP gene regulation, the inventors sought to determine the effects ofincreased and decreased levels of miR-455-3p on cell morphology. Theinventors analyzed the cell morphology after miR-455-3p mimics andinhibitor treatment. Microscopic examination of cells at 24 hrpost-transfection with the miR-455-3p mimics showed extended cellproliferation compared to the controls (FIG. 13C). The cells transfectedwith miR-455-3p inhibitors did not show normal and healthyproliferation. These findings suggest that higher expression levels ofmiR-455-3p mimics promoted cell growth, on the other hand, inhibitorsreduced cell proliferation.

(b) MiR-455-3p and APP expression analysis. To confirm the regulation ofAPP gene by miR-455-3p, cells were transfected with miR-455-3p mimicsand inhibitors, followed by the quantification of miR-455-3p and APPmRNA expression at 24 hr post-transfection. qRT-PCR analysis showed thesignificant upregulation of miR-455-3p (225-fold) (P=0.0001) in cellstransfected with miR-455-3p mimics. On the contrary, cells treated withmiR-455-3p inhibitors showed a significant downregulation (−5.23-fold)of miR-455-3p relative to untreated cells (FIG. 13D).

In the qRT-PCR analysis, it was found that mRNA levels of APP werereduced by 0.3-fold (P=0.001) in the cells transfected with themiR-455-3p mimics. On the other hand, it was found 2.6-fold upregulationof APP expression in the cells transfected with the miR-455-3pinhibitors (FIG. 13E). These results agree with the luciferase assaydata that miR-455-3p had the potential to modulate the APP expression.

(c) Effects of miR-455-3p mimics and inhibitors on cell viability. Asshown in FIGS. 13F and 13G, apoptotic cell death was reduced by 5.31%(P=0.042) in the cells transfected with miR-455-3p mimics relative tountransfected cells. On the contrary, apoptotic cell death was increasedby 10.96% (P=0.0071) in cells transfected with miR-455-3p inhibitors.These findings point to the protective effects of miR-455-3p mimics oncell survival.

Cell viability analysis showed the increased levels of cell survival incells transfected with miR-455-3p mimics compared to the untransfectedcells (FIG. 13H). As expected, cell survival was significantly reducedin cells transfected with miR-455-3p inhibitors. These findings suggestthat miR-455-3p mimics increases cell survival.

Effect of miR-455-3p on mutant APP. To determine the effect of mutantAPP and miR-455-3p on cell survival and cell viability, cells weretransfected with mutant APP cDNA and miR-455-3p vector. Cells wereobserved under a light microscope after 24 hours transfection withmutant APP and miR-455-3p individually and in combination. MiR-455-3pvector had a green fluorescence protein. As shown in FIG. 14A, cellstransfected with miR-455-3p vector showed an increased number of cellsrelative to the miR-control vector (vector without miR-455-3p sequence)and mutant APP cDNA transfected cells.

FIGS. 14A to 14J—Regulation of mutant APP cDNA expression by miR-455-3p.(FIG. 14A) Representative images for transfection of the miR-455-3pexpression vector (GFP), the miR-control (GFP) vector, and the mutantAPP cDNA in neuroblastoma cells (10× magnification). Bright field imagesand green fluorescence detected in the same field at 24 hrpost-transfection (10× magnification). (FIG. 14B) Representative imagesof Annexin V and Propidium Iodide (PI) stained neuroblastoma cellstransfected with the miR-control vector, the miR-455-3p vector, and themutant APP cDNA. The white arrow indicates the populations of viable(green) cells and dead (red) cells. (FIG. 14C) Percentage (%) of anapoptotic cell population after 24 hr of the miR-455-3p vector, themiR-control vector, and the mutant APP cDNA transfecting cells. (FIG.14D) Percentage (%) of a viable cell population after 24 hr of themiR-control vector, the miR-455-3p vector and the mutant APP cDNAtransfecting cells. (FIG. 14E) Quantitative measurement of miR-455-3pfold change expression after 24 hr of the miR-455-3p vector and themutant APP cDNA transfecting cells. (FIG. 14F) Quantitative measurementof APP mRNA folds change expression after 24 hr of the miR-455-3p vectorand the mutant APP cDNA transfecting cells. (FIG. 14G) Western blot forAPP (6E10), the C-terminal fragment of APP (C99) and B-actin proteins incells transfected with the miR-control vector, the miR-455-3p vector,mutant APP cDNA and co-transfected cells. Quantitative measurement of(FIG. 14H) APP (6E10) and C-terminal fragments of APP C99 (FIG. 14I),and C83 (FIG. 14J) proteins levels by densitometry in the mutant APPcDNA and the miR-455-3p vector transfecting cells. (*P<0.05) (**P<0.01)(***P<0.001)

Significantly increased apoptotic cell death was observed in mutant APPcells relative to untransfected cells (18.46%) (P=0.001). Co-transfectedcells with both mutant APP+miR-455-3p showed significantly reducedapoptotic cell death (9.54%) (P=0.031) relative to mutant APP cells(FIGS. 14B and 14C). Cell viability was significantly increased (91.35%)(P=0.014) in cells transfected with miR-455-3p compared to miR-controlvector-transfected cells (71.95%) (FIG. 14D). Similarly, cell viabilitywas higher in cells co-transfected with the mutant APP and miR-455-3pvector compared to cells transfected with mutant APP alone. Thesefindings show that miR-455-3p is protective against mutant APP toxicity.

Effect of miR-455-3p on mRNA and protein levels of mutant APP. Next, theinventors determined the expression of miR-455-3p in miR-control vector,miR-455-3p vector and mutant APP transfected cells and howoverexpression of miR-455-3p modulates the expression of mutant APPcDNA.

The miR-455-3p analysis revealed that increased expression levels ofmiR-455-3p (2285-fold) (P=0.0001) in cells transfected with miR-455-3pvector compared to the miR-control vector (FIG. 14E). The inventors alsoobserved increased levels of miR-455-3p in cells transfected with mutantAPP cDNA compared to miR-control vector (FIG. 14E). As shown in FIG.14F, significantly reduced levels of mutant APP mRNA (1508-fold)(P=0.008) were found in cells co-transfected with miR-455-3p and mutantAPP relative to cells transfected with mutant APP alone.

The immunoblot analysis findings agrees with mRNA data thatsignificantly reduced the level of full-length APP and both C99 and C83fragments in cells co-transfected with the miR-455-3p and mutant APPcells (P=0.0021) relative to mutant APP cells alone (FIGS. 14G, 14H, 14Iand 14J). These observations suggest that miR-455-3p not only modulatefull-length APP but also involved in c-terminal fragments of APP.

Effect of miR-455-3p on the localization of mutant APP. To determine thelocation and immunoreactivity levels of mutant APP and Aβ in cellstransfected with miR-455-3p, mutant APP and in combination,immunostaining of cells were performed. As shown in FIG. 15A, cellstransfected with miR-455-3p, showed green fluorescence protein.Expression intensity of APP (6E10, red) was significantly reduced incells co-transfected with the miR-455-3p and mutant APP cDNA (P=0.027)compared to the cells transfected with mutant APP alone (FIGS. 15A and15B). These findings further confirmed that miR-455-3p has a role inregulating mutant APP and Aβ3 levels.

FIGS. 15A to 15C—MiR-455-3p reduces APP and amyloid proteins. (FIG. 15A)Representative immunostaining images of neuroblastoma cells transfectedwith the miR-control vector, the miR-455-3p vector, the mutant APP cDNA,the mutant APP and miR-455-3p co-transfected. Cells were stain with theAPP (6E10) antibody producing red fluorescence (20× magnification).Green fluorescence showed the miR-control and miR-455-3p vectorexpression and the nucleus of the cell stained with DAPI (blue).Fluorescence intensity (red) of the mutant APP was reduce in mutant APPand miR-455-3p co-transfected cells. (FIG. 15B) Quantitative measurementof fluorescence intensity of the APP (6E10) protein in mutant APP,miR-455-3p, and miR-control vector transfected cells. (FIG. 15C) ELISAanalysis for amyloid ₍₁₋₄₀₎ and ₍₁₋₄₂₎ levels in neuroblastoma cells.Quantitative detection of the (i) human amyloid-β₍₁₋₄₀₎ and (ii)amyloid-β₍₁₋₄₂₎ peptide level in mutant APP cDNA and miR-455-3p vectortransfected cells. (*P<0.05).

Effect of miR-455-3p on Aβ₍₁₋₄₀₎ and Aβ₍₁₋₄₂₎levels. Since, miR-455-3preduced the levels of full-length mutant APP, the levels of solubleAβ₍₁₋₄₀₎ and Aβ₍₁₋₄₂₎ in neuroblastoma cells lysate were checked. Levelof Aβ₍₁₋₄₀₎ was significantly reduced (P=0.011) in the cellsco-transfected with the miR-455-3p and mutant APP compared to cellstransfected with mutant APP alone (FIG. 15C). Similarly, the level ofAβ₍₁₋₄₂₎ was also significantly lowered by miR-455-3p in co-transfectedcells with miR-455-3p and mutant APP compared to cells transfected withmutant APP alone (FIG. 15C). These observations suggest that miR-455-3preduced both Aβ₍₁₋₄₀₎ and Aβ(1-42) levels in mutant APP cells.

Effect of miR-455-3p on mitochondrial biogenesis and synaptic activity.To determine the effect of miR-455-3p on mitochondrial biogenesis andsynaptic proteins, the inventors assessed mitochondrial biogenesis(PGC1a, NRF1, NRF1, TFAM), and synaptic (synaptophysin, and PSD95) genesat mRNA and protein levels.

As shown in Table 9, mRNA levels of mitochondrial biogenesis genesPGC1a, NRF1, NRF1, TFAM were significantly reduced in the mutant APPcells relative to miR-control transfected cells. On the other hand, mRNAlevels of mitochondrial biogenesis genes were significantly increase inco-transfected cells with miR-455-3p and mutant APP relative to cellstransfected with mutant APP alone (Table 9).

TABLE 9 qRT-PCR analysis of neuroblastoma cells transfected with miR-control vector, miR-455-3p vector, mutant APP cDNA and their mRNA foldchange calculations relative to miR-control vector (Foldchange-mRNA/β-actin) miR- Mutant Mutant Mutant 455-3p APP APP + miR-APP + miR- Genes vector cDNA 455-3p control Mitochondrial biogenesisPGC1α 4.24** −1.8*  3.64** −1.7* genes NRF1 3.18** −1.4 2.48* −4.81**NRF2 3.2* −1.59* 2.43* −1.25* TFAM 2.6* −1.4* 2.68* −4.49** Synapticgenes Synaptophysin 2.5* −2.44* 2*    −1.45* PSD95 3.25* −3.29** 2.78*−2.37** Mitochondrial fission genes DRP1 −1.42* 2.36* −1.18    2.43*FIS1 −1.37* 1.89* −1.17    1.61* Mitochondrial fusion genes OPA1 1.59*−1.93* 1.85* −1.4* Mfn1 1.92* −1.46* 1.48* −1.76* Mfn2 2.01* −1.14 1.93*−1.19

Similarly, quantitative densitometry based on immunoblot analysisrevealed decreased protein levels of PGC1α, NRF1, NRF2, and TFAM by themutant APP relative to cells transfected with miR-control. On the otherhand, biogenesis protein levels were increased in miR-455-3p and mutantAPP cDNA co-transfected cells relative to transfected with mutant APPalone, indicating that miR-455-3p enhances biogenesis activity (FIGS.16A-16E).

FIGS. 16A to 16H—Immunoblotting for mitochondrial biogenesis andsynaptic genes. (FIG. 16A) Representative western blot images for PGC1α,NRF1, NRF2, TFAM, and beta-actin proteins levels in 1) Neuroblastomacells transfected with the miR-control vector, 2) Neuroblastoma cellstransfected with the miR-455-3p expression vector, 3) Neuroblastomacells transfected with the mutant APP cDNA, 4) Neuroblastoma cellsco-transfected with the miR-455-3p and mutant APP cDNA, and 5)Neuroblastoma cells co-transfected with the mutant APP and miR-controlvector. Quantitative measurement of the levels of (FIG. 16B) the PGC1αprotein (FIG. 16C) NRF1, (FIG. 16D) NRF2, and (FIG. 16E) TFAM usingdensitometry in 1) Cells transfected with the miR-control vector, 2)Cells transfected with the miR-455-3p expression vector, 3) Cellstransfected with mutant APP cDNA, 4) Cells co-transfected with themiR-455-3p and mutant APP cDNA, and 5) Cells co-transfected with themutant APP and miR-control vector. (FIG. 16F) Representative westernblot images for synaptophysin, PSD95, and beta-actin proteins levelsin 1) Cells transfected with the miR-control vector, 2) Cellstransfected with the miR-455-3p expression vector, 3) Cells transfectedwith the mutant APP cDNA, 4) Cells co-transfected with the miR-455-3pand mutant APP cDNA, and 5) Cells co-transfected with the mutant APP andmiR-control vector. Quantitative measurement of the levels of (G) thesynaptophysin protein and (FIG. 16H) PSD95 using densitometry in samegroups of cells. (*P<0.05) (**P<0.01)

A synaptic protein analysis revealed the significantly reduced levels ofsynaptophysin and PSD95 genes at mRNA (Table 9) and protein levels(FIGS. 16F-16H) in cells transfected with mutant APP relative tocontrols. On the other hand, synaptic protein levels were increasedsignificantly in miR-455-3p co-transfected cells relative to transfectedwith mutant APP alone.

Effect of miR-455-3p on mitochondrial dynamics (fission and fusion).Further, to confirm the effect of miR-455-3p on mitochondrial dynamicsgenes, the inventors studied the expression of mitochondrial fissionproteins (DRP1 and FIS1) and fusion proteins (OPA1, Mfn1, and Mfn2) atmRNA and protein levels. As shown in SI Table 4, mRNA levels of DRP1 andFIS1 were significantly increased in cells transfected with mutant APPcDNA compared to controls. On the contrary, cells co-transfected withmiR-455-3p and mutant APP cDNA showed reduced levels of fission genesDRP1 and FIS1 relative to mutant APP cells (Table 9). As expected, mRNAlevels of mitochondrial fusion genes (OPA1, Mfn1, and Mfn2) weresignificantly reduce in mutant APP cells, but upregulated in the cellsco-transfected with miR-455-3p and mutant APP cDNA (Table 9).

The immunoblotting data agreed with mRNA observations (FIGS. 17A-17F).These results conclude that miR-455-3p reduce fission proteins andenhances fusion proteins.

FIGS. 17A to 17I—Immunoblotting for mitochondrial dynamic genes. (FIG.17A) Representative western blot images for DRP1, FIS1, OPA1, Mfn1, Mfn2and beta-actin proteins levels in: 1) Neuroblastoma cells transfectedwith the miR-control vector, 2) Neuroblastoma cells transfected with themiR-455-3p expression vector, 3) Neuroblastoma cells transfected withthe mutant APP cDNA, 4) Neuroblastoma cells co-transfected with themiR-455-3p and mutant APP cDNA, and 5) Neuroblastoma cellsco-transfected with the mutant APP and miR-control vector. Quantitativemeasurement of the levels of (FIG. 17B) the DRP1, (FIG. 17C) FIS1, (FIG.17D) OPA1, (FIG. 17E) Mfn1 and (FIG. 17F) Mfn2 proteins usingdensitometry in the same groups of cells. (FIG. 17G) Representative TEMimages of Neuroblastoma cells transfected with the miR-control vector,the miR-455-3p vector, mutant APP cDNA, co-transfected with mutant APPand miR-455-3p, and co-transfected with mutant APP and miR-control,showing mitochondrial organization (600 nm magnification). (FIG. 17H)Quantification of the number of mitochondria in the Neuroblastoma cellstransfected with miR-control vector, the miR-455-3p vector, mutant APPcDNA, co-transfected with mutant APP and miR-455-3p, and co-transfectedwith mutant APP and miR-control. (FIG. 17I) Quantification of the sizeof mitochondria (μm) in the neuroblastoma cells transfected withmiR-control vector, the miR-455-3p vector, mutant APP cDNA,co-transfected with mutant APP and miR-455-3p, and co-transfected withmutant APP and miR-control. (*P<0.05) (**P<0.01).

Effect of miR-455-3p on mitochondrial morphology. The inventors alsostudied mitochondrial number and length in mutant APP cells transfectedwith miR-455-3p in order to determine the effect of miR-455-3p onmitochondrial morphology. As shown in FIG. 17G increased numbers ofmitochondria were found in cells transfected with mutant APP cDNArelative to miR-control vector transfected cells, indicating that mutantAPP, fragments mitochondria. However, significantly reduced numbers ofmitochondria were found in cells co-transfected with miR-455-3p andmutant APP relative to cells transfected with mutant APP alone (FIG.17H).

Conversely, the mitochondrial length was decreased in the mutant APPcells, whereas, cells co-transfected with miR-455-3p and mutant APP cDNAshowed increased in mitochondrial length compared to mutant APP cellsalone (FIG. 17I). Taken together, these analyses confirmed thatmiR-455-3p had the potential to control and/or repair the abnormalmitochondrial dynamics caused by toxic effects of mutant APP.

This example targeted miR-455-3p as a therapeutic candidate for personswith AD. The inventors demonstrate the therapeutic use for miR-455-3pthrough the modulation of the APP processing. Additionally, it was foundthat miR-455-3p promote cell survival and activates synaptic genes, alsoindicating that miR-455-3p may have a protective role in AD pathogenesisthrough the modulation of defective mitochondrial biogenesis andmitochondrial dynamics.

The bioinformatics analysis showed that miR-455-3p targets several othergenes (e.g., BAX, BMF, TGFBR3L, and HIF1 [http://targetscan.org]) thathave important roles in apoptotic pathways. However, in current study, afocus was on the regulation of APP processing by miR-455-3p, and how itovercomes the toxic effect of mutant APP on cell survival, mitochondrialbiogenesis, mitochondrial dynamics, and synaptic activity.

The two constitutive binding sites of miR-455-3p at the 3′UTR region ofthe APP gene provides better regulation and modulation of miRNAmoieties. Overexpression of miR-455-3p in mutant APP cells,significantly reduced the levels of mutant full-length APP and Aβ₍₁₋₄₀₎& Aβ₍₁₋₄₂₎. These findings strongly suggest that miR-455-3p is apotential candidate to treat AD. In this study, the inventors used amutant APP cDNA construct that robustly expressed the human mutant APPprotein in HT22 cells [32]. The inventors found that an overexpressionof mutant APP, causes mitochondrial, synaptic, and autophagy/mitophagyabnormalities in hippocampal neurons, in addition to reduced cellsurvival, leading to neuronal dysfunction [32]. mRNA levels of APP weresignificantly increased (2745-fold) in mutant APP cDNA transfectedcells. Despite this huge increase, miR-455-3p was capable of efficientlysuppress the toxic effect of mutant APP on cell survival, mitochondrialbiogenesis/dynamics, and synaptic activity. It was also found thatmiR-455-3p not only interact and modulate full-length APP levels, butalso suppress C99 fragment of APP, which is responsible for thegeneration of Aβ₍₁₋₄₀₎ and Aβ₍₁₋₄₂₎ peptides [39].

The inventors also identified a supportive role of miR-455-3p inmitochondrial biogenesis, consistent with Zhang et al. (2015), whoreported that miR-455 regulates brown adipogenesis via a novelHIF1an-AMPK-PGC1α signaling network [40]. The inventors confirmed thatoverexpression of the mutant APP cDNA caused the suppression of PGC1αand other mitochondrial biogenesis genes. In this example,over-expression of miR-455-3p not only increased PGC1α but alsoincreased the expression of their downstream effector genes NRF-1,NRF-2, and TFAM. Therefore, upregulation of mitochondrial biogenesisevents eventually improved the overall mitochondrial functions. Thesefindings are supported by the inventors' electron microscopic analysisof neuroblastoma cells transfected with mutant APP cDNA and miR-455-3p,miR-455-3p was associated with an increase in mitochondrial length and adecrease in the number of mitochondria number. Further, analysis ofmitochondrial dynamics proteins showed the suppression of fissionprotein (DRP1 and FIS1) and upregulation of fusion proteins (OPA1, Mfn1,and Mfn2) by miR-455-3p. Based on these observations, it was found thatoverexpression of miR-455-3p maintain the normal mitochondrial activityby reducing the toxic effects of Aβ on mitochondria.

Additionally, the upregulation of the key synaptic genes synaptophysinand PSD95 in presence of miR-455-3p provides new evidence thatmiR-455-3p has a role in the regulation of synaptic activity. Theimproved cell survival and extended dendrites in miR-455-3poverexpressing cells, further supports its possible role in the neuronalproliferation. This example is the first to describe that miR-455-3p hassignificant role progression and pathogenesis in AD.

To understand the impact of miR-455-3p in the brain, overexpressed(transgenic) and/or (depleted) knockout mouse models for miR-455-3p canbe characterized. Further, transgenic and knockout lines of miR-455-3pmice with different mutant APP mice (APP-Tg2576), APP/PS1, 5XFAD andothers) and study abnormal APP processing, C-terminal fragments such asC83 & C99, Aβ levels and mitochondrial biogenesis, mitochondrialdynamics and synaptic activities in double mutant mice (Tg miR455-3p×APPTg mice; KO miR455-3p×APP Tg mice) relative to APP Tg mice alone can bestudied.

In summary, miR-455-3p increases cell survival and promotes cellproliferation, and overexpression of miR-455-3p dramatically decreasesfull-length APP expression, the C-terminal fragment of APP and reducesAβ₍₁₋₄₀₎ and Aβ₍₁₋₄₂₎ levels. Further, increased levels of miR-455-3palso increase mitochondrial biogenesis, mitochondrial fusion, andsynaptic genes. These findings suggest the negative role of miR-455-3pin abnormal APP processing and its positive role in the regulation ofmitochondrial biogenesis/dynamics and synaptic plasticity make it avaluable potential molecule worthy of research to identify AD therapy.

Example 4

MiRNAs are present in small, subcellular compartments of the neuron suchas neural dendrites, synaptic vesicles, and synaptosomes are known assynaptic miRNAs. Synaptic miRNAs involved in governing multiple synapticfunctions that lead to healthy brain functioning and synaptic activity.However, the precise role of synaptic miRNAs had not previously beendetermined in AD progression. This example emphasizes the presence ofsmall RNAs and miRNAs at the synapse, synaptic vesicles, andsynaptosomes. The inventors summarize some potential synaptic miRNAs,their functions, and how these synaptic miRNAs regulate the levels ofsynaptic proteins locally at the synapse. Finally, the impact ofsynaptic miRNAs in AD progression concerning the synaptic ATPproduction, mitochondrial function, and synaptic activity is discussed.

The purpose of this example was to assess the role of functional anddysfunctional synapse-associated miRNAs in AD progression. The inventorslooked into the presence of small RNAs and miRNAs at the synapse, thesynaptic vesicles, and synaptosomes and accessed their roles inregulating synaptic activity in AD and other neurodegenerative diseases.In this example, the inventors provide results that has focused onsynaptic miRNAs as possible therapeutic agents that capable ofpreventing or delaying AD onset or progression.

The presence of small RNAs populations was discovered in the synapticvesicles (SVs). SVs are small neuronal presynaptic organelles that carrythe neurotransmitters and release them to the synaptic cleft. Before, itwas presumed that synaptic vesicles contain the neurotransmitters only.Li et al (2015), identified the presence of small ribonucleic acids(sRNAs), in the synaptic vesicles isolated from the electric organ ofTorpedo californica in addition to the regular neurotransmitters [17].Most of the sRNAs are transfer RNA molecules termed as tRNA fragments[17]. Not even Torpedo californica, but the SVs of the mouse braincontain abundant levels of sRNAs, including transfer RNA fragments(trfRNAs) and miRNAs [17]. These findings show that SV sRNAs areconserved and are possibly involved in transcriptional and translationalregulation of local synaptic proteins. Besides, the release ofneurotransmitters, now it is exciting to know whether SV contained sRNAsregulate the synaptic activity or change dendritic excitability. The SVsspecific trfRNAs and miRNAs, both could directly regulate local proteinsynthesis and could have broad implications on neurotransmission andsynaptic activity.

MiR-134 and miR-138 are the two potent neural miRNAs that playedimportant roles in synapse development and synaptic activity. MiRNAspresent throughout the cells, and some miRNAs are localized to cellularorganelles. Subcellular compartmentalization and localization of miRNAs,microRNA induced silencing complex, and target mRNA have been observedto localize in multiple subcellular compartments, including roughendoplasmic reticulum, P-bodies, stress granules in the trans-Golginetwork, early and late endosomes, multivesicular bodies, lysosomes,mitochondria, and the nucleus.

The synaptic vesicles extracted from mouse CNS that contains severalsmall RNAs, transfer-RNA, and miRNAs (miR-128-1, miR-99a, miR-100,miR-22, and miR-127) included high copy numbers of miR-128-1 and miR-99arelative to other miRNAs. The most abundant miRNA, miR-128-1, isimportant for neuronal development, synaptogenesis, and post-mitoticneuronal functioning. The members of the miR-99 family (miR-99a) havebeen shown to co-enrich with polyribosomes in mammalian neurons and toregulate the mammalian target of the rapamycin (mTOR) pathway. Otherthan miR-128-1 and miR-99a, several other miRNAs (miR-124a-3p,miR-136-5p, and miR-376a-3p) were also abundantly expressed withinsynaptoneurosomes isolated from the prion-infected forebrain of animalmodel. These synaptoneurosomes associated or synaptosome-specific miRNAs(termed synapto-miRs) could be very important in AD research and otherneurodegenerative diseases. The potential synaptic miRNAs and their mainfunctions are summarized in Table 10.

TABLE 10 Summary of synaptic miRNAs, their location and molecularfunctions. Synaptic miRNAs Location Functions Target genes ReferencesMiR-134 Synaptodendritic Synaptic development, Limk1 [59] compartmentssynaptic maturation, and synaptic plasticity MiR-138 Brain Local proteintranslation at Lypla1 [62] the synapse and synaptic plasticity. MiR-146and MiR- Synaptic fraction Synaptic plasticity [15] 125a MiR-125aSynaptosome Suppression and/or P5D95 [16] degradation of target mRNAs atlocal synapses MiR-128-1, Neuron (Synaptic Regulates motor behaviorextracellular [17, 63] vesicles) by modulating neuronal signal-regulatedsignaling networks and kinase ERK2 excitability. network’ MiR-99aPolyribosomes in Regulate the mammalian [17, 64] mammalian neuronstarget of the rapamycin (mTOR) pathway MiR-338 Brain Mitochondrialactivity and COXIV [79] ATP production MiR-151a-5p Cellular respirationand Cytochrome b [81] ATP production (Cytb) MiR-423-3p ATP productionand Cox6a2 [82] energy metabolism MiR-125b Brain Synaptic deficits,Synapsin-2 (SYN- [83] neurotrophic deficits, and 2) and 15- astrogliosislipoxygenase (15- LOX) MiR-124 Brain Synaptic dysfunction and PTPN1 [19]memory loss in AD MiR-30b Synaptic integrity in AD Ephrin type-B [20]receptor 2 (ephB2), Sirtuin1 (sirt1), and Glutamate ionotropic receptorAMPA type subunit 2 (GluA2).

The role of miRNAs has been determined in the regulation of synapticactivity in AD and other neurodegenerative diseases. MiRNA plays animportant role in regulating synaptic plasticity, includingsynaptogenesis, alteration of synaptic morphology, and modification ofsynaptic functions. However, the function of synapse enriched miRNAs isnot determined in great detail in AD, although miRNAs, enriched atsynaptic compartments, directly regulate local protein synthesis(synaptic proteins) and play a crucial role in neurotransmission. For along time, synaptosomes were prepared from the postmortem brains of ADpatients and studied for AD-associated deficits such asneurotransmission, including acetylcholine, glutamate, andc-aminobutyric acid (GABA) systems. These synaptosome studies identifieddecreased levels of neprilysin in AD patients. In other studies,synaptosomes were prepared from the frontal cortical region of ADpostmortem brains to study Aβ aggregates in a synaptosomal fraction.Therefore, the study on the synaptosomal miRNAs was needed in AD.

FIG. 18 shows a proposed mechanism of action mt miRNAs on themitochondrial function, ATP production, and synaptic activity, whichmechanism is not a limitation of the present invention. FIG. 18 is apictorial representation of miRNAs action on mitochondrial function andATP production in the neuron. Healthy neurons showed the normal synapticactivity and neurotransmission, whereas in diseases state such as AD,Aβ, and tau toxicities cause miRNAs alteration in the neuron. Increasedlevel of miRNAs reduces their target gene expression, those areimportant for mitochondrial function. Reduction of mitochondrialproteins leads to impaired mitochondrial function, reduced ATP, and poorsynaptic activity.

Although definitive proof that miRNAs function in a localized manner inthe neurons is still lacking, it is quite possible that miRNAs localizedin dendrites and axon terminals could modulate the expression of localmRNAs and proteins. As shown in FIG. 19, the positive and negativeregulation of proteins at the synapse by local miRNAs could altersynaptic functions. In a healthy state, cell homeostasis maintainsbalanced miRNAs levels, where miRNAs function in a positive way tomaintain healthy cells functioning, e.g., miR-128 regulates the ERK2pathway [63]. Healthy neurons showed normal synaptic activity andneurotransmission. In AD state, the deregulation of synaptic miRNAscaused by the Aβ and tau toxicities. Increased level of miRNAs-miR-124,miR-125b, and miR-30b leads to the downregulation of their targetgene/proteins (PTPN1, SYN-2, 15-LOX, EphB2, Sirt1, and GluA2) which areimportant for synaptic plasticity [19,20,83]. Suppression of synapticand other proteins could impair synapse activity and neurotransmission(FIG. 19).

FIG. 19 is a working model of synaptic miRNAs: miRNAs localized indendrites and axon terminals could modulate the expression of localmRNAs and proteins. Positive and negative regulation of synapticproteins by local miRNAs could alter synaptic function. Neurons notaffected by AD (healthy neuron) showed balanced miRNAs levels and normalsynaptic activity and synaptic neurotransmission. Brain-specific miR-128showed the positive modulation of synaptic activity via the ERK2pathway. On the other hand in AD state, the deregulation of thebrain-specific miRNAs (miR-124 and miR-125b) and other miR-30bnegatively modulate the synaptic activity via suppression of theirtarget genes. The suppression of synaptic and other proteins leads toimpaired synaptic activity and neurotransmission.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In embodiments of any of the compositions andmethods provided herein, “comprising” may be replaced with “consistingessentially of” or “consisting of”. As used herein, the phrase“consisting essentially of” requires the specified integer(s) or stepsas well as those that do not materially affect the character or functionof the claimed invention. As used herein, the term “consisting” is usedto indicate the presence of the recited integer (e.g., a feature, anelement, a characteristic, a property, a method/process step or alimitation) or group of integers (e.g., feature(s), element(s),characteristic(s), property(ies), method/process steps or limitation(s))only.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skill in the art recognize themodified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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What is claimed is:
 1. A method of protecting neuronal cells fromAβ-induced toxicities in a subject comprising: providing the subject inneed of treatment for an Aβ-induced toxicity with an agent thatincreases the miRNA-445-3p in the subject, wherein an increase inmiR-455-3p expression in the neuronal cells enhances neuronal cellsurvival.
 2. The method of claim 1, wherein the agent that increases themiRNA-445-3p in the subject is a nucleic acid vector that expressesmiRNA-445-3p in neuronal cells of the subject.
 3. The method of claim 1,wherein the agent enhances at least one of: mitochondrial biogenesis,enhances synaptic activity or mitochondrial health.
 4. The method ofclaim 1, further comprising providing the subject with a cyclooxygenaseinhibitor, a Catecholamine transferase inhibitor, a protein kinasesinhibitor, a Neurotransmitter transporter inhibitor, a Renin-angiotensinsystem inhibitor, a EGFR tyrosine kinase inhibitor, or a HMG-CoAreductase inhibitor.
 5. The method of claim 1, further comprising thestep of identifying a subject in need of treatment for the Aβ-inducedtoxicity by detecting the presence or the increase in miRNA-445-3p toproduce a score that is indicative of a likelihood of developing AD,wherein a higher score relative to a healthy control indicates that thepatient is likely to have the prognosis for transitioning to classifiedAD, wherein the healthy control is derived from a non-AD patient with noclinical evidence of AD.
 6. The method of claim 5, wherein the patientis identified at least 0.1, 0.9, 2.0, 3.5, or greater than 3.5 yearsprior to reaching clinical disease classification.
 7. The method ofclaim 5, wherein the step of assessing comprises RT-PCR, qRT-PCR,biochip, singleplexed or multiplexed RT-PCR.
 8. The method of claim 5,further comprising assessing at least one additional biomarker selectedfrom: hsa-miR-3613-3p and hsa-miR-4668-5p, which is up-regulated.
 9. Themethod of claim 5, further comprising assessing at least one additionalbiomarker selected from: hsa-mir-320d-2, hsa-miR-378h, hsa-miR-3921,hsa-miR-6805-5p, hsa-miR-92a-3p, hsa-miR-3613-5p, which isdown-regulated.
 10. The method of claim 5, wherein obtaining the datasetassociated with the sample comprises obtaining the sample and processingthe sample to experimentally determine the dataset, or wherein obtainingthe dataset associated with the sample comprises receiving the datasetfrom a third party that has processed the sample to experimentallydetermine the dataset.
 11. The method of claim 5, further comprisingidentifying a relative of the patient at risk for AD by obtaining ascore from a dataset associated with a blood, serum, or plasma samplefrom a relative of the AD patient prior to reaching clinical diseaseclassification.
 12. The method of claim 5, wherein the healthy controlis a pre-determined average level derived from a healthy individual withno clinically documented evidence of AD.
 13. The method of claim 5,wherein at least one of: the miR-455-3p has a greater that 20-foldincrease in expression in Braak stage V and VI compared to controls; theexpression of miR-3613-3p is higher in the brain tissues at Braak stageV when compared to controls; the expression of miR-4668-5p up-regulatedin AD brains at Braak stages IV, V, and VI, but less than miR-455-3p orMiR-4674; the expression of mir-6722 was down-regulated in AD serumsamples Braak stages I to III, but increased in the AD patients at Braakstage VI, V, and VI; the upregulation of miR-455-3p was significantlyhigher in postmortem brains from AD patients at Braak stage V having anApoE (3/4) genotype when compared to controls; or the Braak stage can bedifferentiated between Stage I to III versus Brask Stage IV to VI bycomparing the expression of miR-455-3p, miR-3613-3p, miR-4668-5p, andmir-6722.
 14. A method for treating at least one of amyloid precursorprotein (APP) processing, amyloid beta (Aβ) formation, defectivemitochondrial biogenesis/dynamics and synaptic damage in AD progressionin a subject suspected of having Alzheimer's disease (AD) comprising:providing the subject in need of treatment for an Aβ-induced toxicitywith a vector increases the expression of miRNA-445-3p in the subject,wherein an increase in miR-455-3p expression in the neuronal cellsenhances neuronal cell survival, wherein the amount is sufficient to atleast one of correct amyloid precursor protein (APP) processing, preventamyloid beta (Aβ) formation, decrease defective mitochondrialbiogenesis/dynamics or reduce or prevent synaptic damage in ADprogression.
 15. The method of claim 14, wherein the expression ofmiRNA-445-3p in neuronal cells reduces a level of mutant APP, Aβ(1-40)and Aβ(1-42), and C99 by miR-455-3p.
 16. The method of claim 14, furthercomprising administering a cyclooxygenase inhibitor, a Catecholaminetransferase inhibitor, a protein kinases inhibitor, a Neurotransmittertransporter inhibitor, a Renin-angiotensin system inhibitor, a EGFRtyrosine kinase inhibitor, or a HMG-CoA reductase inhibitor.
 17. Themethod of claim 14, further comprising assessing at least one additionalbiomarker selected from: hsa-miR-3613-3p and hsa-miR-4668-5p, which isup-regulated.
 18. The method of claim 14, further comprising assessingat least one additional biomarker selected from: hsa-mir-320d-2,hsa-miR-378h, hsa-miR-3921, hsa-miR-6805-5p, hsa-miR-92a-3p,hsa-miR-3613-5p, which is down-regulated.
 19. The method of claim 14,wherein the patient is identified at least 0.1, 0.9, 2.0, 3.5, orgreater than 3.5 years prior to reaching clinical diseaseclassification.
 20. The method of claim 14, wherein the step ofassessing comprises RT-PCR, qRT-PCR, biochip, singleplexed ormultiplexed RT-PCR.
 21. A method for treating a subject with Alzheimer'sDisease (AD) comprising: obtaining a blood, serum, or plasma sample fromthe AD patient; assessing the dataset for a presence or an increase inan amount of miRNA-445-3p; and if the subject has a decrease inmiRNA-445-3p expression or is in need of an increase in the expressionof miRNA-445-3p, providing the subject in need of treatment for anAβ-induced toxicity an agent that increases the miRNA-445-3p in thesubject, wherein an increase in miR-455-3p expression in the neuronalcells enhances neuronal cell survival.
 22. The method of claim 21,further comprising administering a cyclooxygenase inhibitor, aCatecholamine transferase inhibitor, a protein kinases inhibitor, aNeurotransmitter transporter inhibitor, a Renin-angiotensin systeminhibitor, a EGFR tyrosine kinase inhibitor, or a HMG-CoA reductaseinhibitor.
 23. The method of claim 21, wherein the step of assessingcomprises RT-PCR, qRT-PCR, biochip, singleplexed or multiplexed RT-PCR.24. The method of claim 21, further comprising assessing at least oneadditional biomarker selected from: hsa-miR-3613-3p and hsa-miR-4668-5p,which is up-regulated.
 25. The method of claim 21, further comprisingassessing at least one additional biomarker selected from:hsa-mir-320d-2, hsa-miR-378h, hsa-miR-3921, hsa-miR-6805-5p,hsa-miR-92a-3p, hsa-miR-3613-5p, which is down-regulated.
 26. The methodof claim 21, wherein obtaining the dataset associated with the samplecomprises obtaining the sample and processing the sample toexperimentally determine the dataset, or wherein obtaining the datasetassociated with the sample comprises receiving the dataset from a thirdparty that has processed the sample to experimentally determine thedataset.
 27. The method of claim 21, further comprising identifying arelative of the patient at risk for AD by obtaining a score from adataset associated with a blood, serum, or plasma sample from a relativeof the AD patient prior to reaching clinical disease classification. 28.The method of claim 21, wherein the healthy control is a pre-determinedaverage level derived from a healthy individual with no clinicallydocumented evidence of AD.
 29. A composition comprising a recombinantanti-miRNA-445-3p nucleic acid sufficient to knockdown the expression ofmiRNA-445-3p.
 30. A method of treating a subject in need of therapy forAlzheimer's Disease comprising: identifying a subject suspected ofhaving Alzheimer's Disease; and providing the subject with an effectiveamount of a recombinant miRNA-445-3p expression vector sufficient toupregulate the expression of miRNA-445-3p in the subject in need oftherapy for Alzheimer's Disease.